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| United States Patent |
5,310,874
|
|
Tamura
, et al.
|
May 10, 1994
|
Integrin .alpha. subunit cytoplasmic domain polypeptides and antibodies
Abstract
Diagnostic systems, methods, polypeptides and antibodies for detecting the
presence of the cytoplasmic domain of the integrin .alpha..sub.6B or
.alpha..sub.3B subunit in a body sample are disclosed
| Inventors:
|
Tamura; Richard N. (San Diego, CA);
Quaranta; Vito (La Jolla, CA)
|
| Assignee:
|
The Scripps Research Institute (La Jolla, CA)
|
| Appl. No.:
|
695564 |
| Filed:
|
May 3, 1991 |
| Current U.S. Class: |
530/324; 436/536; 530/325; 530/326; 530/387.9; 530/388.2 |
| Intern'l Class: |
C07K 007/00; C07K 015/28 |
| Field of Search: |
530/324-326,350,388.2,387.9
514/13
|
References Cited
Other References
Hemler et al. "Association of the VLA .alpha..sup.6 Subunit with a Novel
Protein" J. Biol. Chemistry 264(11) 6529-6535, Apr. 1989.
|
Primary Examiner: Hill, Jr.; Robert J.
Assistant Examiner: Spector; L.
Attorney, Agent or Firm: Bingham; Douglas A., Fitting; Thomas, Logan; April C.
Claims
What is claimed is:
1. An antibody molecule that immunoreacts with a polypeptide consisting of
an amino acid residue sequence shown in SEQ ID NO 3 from residue
1068-1091.
2. The antibody molecule of claim 1 wherein said antibody molecule is a
monoclonal antibody molecule.
3. An antibody molecule that immunoreacts with a polypeptide consisting of
an amino acid residue sequence shown in SEQ ID NO 3 from residue 1045 to
residue 1091.
4. The antibody molecule of claim 3 wherein said antibody molecule is a
monoclonal antibody molecule.
5. An antibody molecule that immunoreacts with a polypeptide consisting of
an amino acid residue sequence shown in SEQ ID NO 5 from residue 121 to
residue 141.
6. The antibody molecule of claim 5 wherein said antibody molecule is a
monoclonal antibody molecule.
7. An antibody molecule that immunoreacts with a polypeptide consisting of
an amino acid residue sequence shown in SEQ ID NO 9 from residue 113 to
residue 153.
8. The antibody molecule of claim 7 wherein said antibody molecule is a
monoclonal antibody molecule.
9. A method for detecting the presence of antigen having the cytoplasmic
domain of .alpha..sub.6B in a body sample comprising the steps of:
a) admixing the body sample with a composition containing antibody
molecules that immunoreact with the .alpha..sub.6B protein and with a
polypeptide consisting essentially of an amino acid residue sequence shown
in SEQ ID NO 3 from residue 1045 to residue 1091 to form an immunoreaction
admixture;
b) maintaining said immunoreaction admixture under immunoreaction
conditions for a time period sufficient for said antibody molecules to
immunoreact with any .alpha..sub.6B present in said body sample and form
an immunoreaction complex; and
c) detecting the presence of any immunoreaction complex formed in step (b)
and thereby detecting the presence of said antigen in said body sample.
10. An isolated polypeptide comprising the .alpha..sub.6B cytoplasmic
domain sequence which consists essentially of an amino acid residue
sequence shown in SEQ ID NO 5 from residue 121 to residue 141.
11. An isolated polypeptide that comprising the .alpha..sub.3B cytoplasmic
domain sequence which consists essentially of an amino acid residue
sequence shown in SEQ ID NO 9 from residue 113 to residue 153.
Description
TECHNICAL FIELD
The present invention relates to polypeptides that define the integrin
.alpha..sub.6 and .alpha..sub.3 subunits, particularly the cytoplasmic
domain of the .alpha..sub.6 and .alpha..sub.3 subunits. In addition, the
invention describes antibodies immunoreactive with the cytoplasmic domain
of .alpha..sub.6 and .alpha..sub.3, and methods for using the antibodies
and polypeptides in assays for detecting .alpha..sub.6 and .alpha..sub.3
subunits in body samples.
BACKGROUND
The integrin family of cell surface receptors serve cellular adhesion
functions. The receptors form a link between the extracellular matrix and
the cytoskeleton through their binding to various extracellular
components. Each integrin receptor is a heterodimer comprised of an
.alpha. and a .beta. subunit. At least 11 .alpha. chains (Ruoslahti and
Giancotti, 1989) and six .beta. chains (Sheppard et al., 1990) have been
recognized in man. Each .alpha. subunit tends to associate with only one
type of .beta. subunit, but there are several exceptions to this rule
(Hemler et al., 1989; Cheresh et al., 1989; Holzmann et al., 1989; Freed
et al., 1989).
The human heterodimer VLA-6 was identified using the monoclonal antibody
GoH3, which is immunoreactive with the .alpha..sub.6 subunit expressed on
the surface of mouse and human cells. Hemler et al. J. Biol. Chem.,
263:7660-7665, (1988); and Sonnenberg et al. J. Biol. Chem.,
262:10376-10383, (1987). The amino terminal sequence of the human VLA-6
.alpha..sub.6 subunit was determined from purified protein (Kajiji et al.
EMBO J. 8:673-680,1989) and was used to design degenerate oligonucleotides
for probing a cDNA library. The full length sequence of .alpha..sub.6
cDNA, and its predicted amino acid sequence, were elucidated subsequent to
cDNA cloning. Tamura, et al., J. Cell Biol., 111:1593-1604 (1990). While
Tamura et al., supra, also disclose multiple cDNA sequences encoding the
VLA-6 .beta..sub.4 subunit, there is provided no evidence that additional
VLA-6 .alpha..sub.6 subunits exist. European Patent Application
Publication Number 279,669 (published Jul. 24, 1988) describes human
.alpha..sub.6 and .beta..sub.4 subunits of an integrin receptor and the
complex they associate to form on pancreatic and other cancer cells. The
publication does not describe or suggest that an isoform of the
.alpha..sub.6 subunit exits.
The full length sequence of a hamster cDNA encoding the Gap b3 cell surface
membrane glycoprotein was described by Tsuji et al., J. Biol. Chem.,
265:7016-7021 (1990). Based on the predicted amino acid sequence and
predicted overall structure, it was suggested that Gap b3 is the hamster
homolog of the .alpha..sub.3 integrin subunit. The sequence of a cDNA
encoding the partial sequence of chicken .alpha..sub.3 protein was
disclosed in Hynes et al. J. Cell Biol., 109:409-420 (1989). The
cytoplasmic regions of these clones do not share homology with the
cytoplasmic region of .alpha..sub.3B disclosed herein, and are therefore
assumed to encode .alpha..sub.3A subunit isoform. Furthermore, neither
publication suggest the possibility of an .alpha..sub.3B subunit.
The N-terminal amino acid sequence of human .alpha..sub.3 protein is
provided in European Patent Application Publication Number 330,506
(published Jul. 3, 1989). That publication provides no suggestion that an
isoform of the .alpha..sub.3 protein, namely .alpha..sub.3B, exists.
BRIEF SUMMARY OF THE INVENTION
A new species of alpha (.alpha.) integrin subunit protein has been
discovered, with representative members in both the .alpha..sub.6 and
.alpha..sub.3 class of integrins corresponding to the laminin receptor and
the laminin, collagen and fibronectin receptors, respectively.
Specifically, it has been discovered that new .alpha..sub.6 species and
.alpha..sub.3 species exist which differ from previously described
.alpha..sub.6 and .alpha..sub.3 proteins in the cytoplasmic domain of the
protein. Through a combination of cDNA sequencing studies and
anti-synthetic peptide antibody immunoreactivity studies, it has been
shown that the cytoplasmic domain of these new proteins, designated
.alpha..sub.6B and .alpha..sub.3B, are related between human and mouse
isolates.
Thus the present invention describes polypeptides comprising an amino acid
residue sequence that includes the amino acid residue sequence defining an
antigenic determinant in the cytoplasmic domain of the human or mouse
.alpha..sub.6B or .alpha..sub.3B protein. Preferably the polypeptide has a
sequence corresponding to the whole cytoplasmic domain of either the human
or mouse .alpha..sub.6B or .alpha..sub.3B protein. Alternatively, a
polypeptide can correspond to all or substantially all of a native human
or mouse .alpha..sub.6B or .alpha..sub.3B subunit in substantially
isolated form.
The polypeptides or proteins are useful as immunogens for preparing
polyclonal and monoclonal antibodies immunoreactive with the human or
mouse .alpha..sub.6B or .alpha..sub.3B cytoplasmic domains, and as
reagents for use in diagnostic assays for detecting the .alpha..sub.6B or
.alpha..sub.3B proteins.
Thus, in a related embodiment the invention describes polyclonal and
monoclonal antibodies having imunospecificities for antigenic determinants
on the cytoplasmic domains of .alpha..sub.6B and .alpha..sub.3B proteins.
These antibodies find use in in vitro and in situ immunoassays for
detecting .alpha..sub.6B or .alpha..sub.3B cytoplasmic domain antigens in
body samples such as tissues or fluids.
Another aspect of the invention is the diagnostic methods and kits
therefor, for detecting .alpha..sub.6B or .alpha..sub.3B cytoplasmic
domain antigenic determinants using an antibody of this invention.
Other features and benefits of the invention will become apparent from the
following detailed description and specific examples describing the
invention, its principles and preferred embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings forming a portion of this disclosure:
FIGS. 1A and 1B illustrates immunoprecipitation of polypeptides from mouse
cells using antibodies specific for the .alpha..sub.6 subunit. The
differentiated (Diff.) ES1 and D3 cells are described in Example 2.
Antibody GoH3 is a monoclonal antibody immunospecific for the
extracellular domain of the .alpha..sub.6A subunit. Antisera 6844 was
raised in rabbit against a synthetic peptide specific for the cytoplasmic
domain of human .alpha..sub.6A. The immunoprecipitated labeled proteins
were visualized by SDS-PAGE. Molecular weight, in kilodaltons, is noted on
the side of the gel.
FIGS. 2 and 3 illustrate a sequential immunoprecipitation analysis of
.alpha..sub.6 subunits in human JAR cell lysates as described in Example
2. NRS is normal rabbit preimmune sera, anti-.alpha..sub.6 Mab is GoH3,
anti-.alpha..sub.6A is sera 6844 and anti-.alpha..sub.6B is sera 382. The
molecular weight of standard protein markers is shown on the right side of
the gel and is expressed in kilodaltons (KDa). FIG. 2 shows
immunodepletion with NRS or with anit-.alpha..sub.6 Mab, and FIG. 3 shows
immunodepletion with anti-.alpha..sub.6A or with anti-.alpha..sub.6B.
FIG. 4 shows .alpha..sub.6A and .alpha..sub.6B PCR amplification products
visualized on an ethidium stained gel. Single-stranded cDNA was generated
from human PG, JAR and U937 cells and was amplified with a set of primers,
1156 and 1157, specific for the human .alpha..sub.6A sequence as described
in Example 3. The primers were also used to amplify the cloned human
.alpha..sub.6A cDNA sequence, which yielded an amplification product of
about 540 bp. The amplification products from the tested cell lines were
either 540 bp or 410 bp, or both.
FIG. 5 compares the nucleotide sequences of the 540 bp and 410 bp
amplification products described in FIG. 4. The 540 bp product shown on
the top line is designated .alpha..sub.6A, and the 410 bp product shown on
the bottom line is designated .alpha..sub.6B. Vertical bars denote where
the two sequences are homologous. Horizontal dots denote a 130 nucleotide
(nt) deletion in the .alpha..sub.6B sequence with respect to the
.alpha..sub.6A sequence. The 130 nt deletion is in the region that encodes
the .alpha..sub.6A cytoplasmic domain.
FIG. 6 provides and compares the predicted amino acid sequence for the
.alpha..sub.6 amplification products shown in FIG. 5. The solid arrows
show the location of the outer PCR primers; the broken arrows show the
location of the nested inner PCR primers. The underlined sequence
represent the putative transmembrane domain. The open boxed area is the
.alpha..sub.6A cytoplasmic domain; the shaded boxed area is the
.alpha..sub.6B cytoplasmic domain. The bracketed area represents the 130
nt sequence deleted from the .alpha..sub.6B sequence.
FIG. 7A and 7B depicts an ethidium bromide-stained gel of the PCR
amplification products generated from (A) undifferentiated ES1 and B16
cells and (B) undifferentiated and differentiated ES1 cells as described
in Example 3. The same priming oligonucleotides were used to amplify cDNA
from these cells.
FIG. 8 provides and compares the nucleotide and predicted amino acid
sequences for the mouse .alpha..sub.6 amplification products shown in FIG.
7. The .alpha..sub.6B sequence is on the top line; the .alpha..sub.6A
sequence is on the bottom line. Predicted amino acid residues are noted
below the nucleotide sequence. The solid arrows show the location of the
PCR primers. The boxed regions encompass the start of cytoplasmic domain
for the .alpha..sub.6A and .alpha..sub.6B proteins, respectively.
FIGS. 9A and 9B illustrates the results of in situ immunostaining of
diseased human kidney tissue. Panel A is stained with polyclonal antisera
6488 specific for the .alpha..sub.6A cytoplasmic region. Panel B is
stained with polyclonal antisera 382 specific for the .alpha..sub.6B
cytoplasmic region.
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
Amino Acid Residue: An amino acid formed upon chemical digestion
(hydrolysis) of a polypeptide at its peptide linkages. The amino acid
residues described herein are preferred to be in the "L" isomeric form.
However, residues in the "D" isomeric form can be substituted for any
L-amino acid residue, as long as the desired functional property is
retained by the polypeptide. NH.sub.2 refers to the free amino group
present at the amino terminus of a polypeptide. C00H refers to the free
carboxy group present at the carboxy terminus of a polypeptide. In keeping
with standard polypeptide nomenclature described in J. Biol. Chem.,
243:3552-59 (1969) and adopted at 37 C.F.R. 1.822(b) (2)), abbreviations
for amino acid residues are shown in the following Table of
Correspondence:
______________________________________
TABLE OF CORRESPONDENCE
SYMBOL
1-Letter 3-Letter AMINO ACID
______________________________________
Y Tyr tyrosine
G Gly glycine
F Phe phenylalanine
M Met methionine
A Ala alanine
S Ser serine
I Ile isoleucine
L Leu leucine
T Thr threonine
V Val valine
P Pro proline
K Lys lysine
H His histidine
Q Gln glutamine
E Glu glutamic acid
W Trp tryptophan
R Arg arginine
D Asp aspartic acid
N Asn asparagine
C Cys cysteine
______________________________________
It should be noted that all amino acid residue sequences are represented
herein by formulae whose left and right orientation is in the conventional
direction of amino-terminus to carboxy-terminus. In addition, the phrase
"amino acid residue" is broadly defined to include the amino acids listed
in the Table of Correspondence and modified and unusual amino acids, such
as those listed in 37 C.F.R. 1.822(b)(4), and incorporated herein by
reference. Furthermore, it should be noted that a dash at the beginning or
end of an amino acid residue sequence indicates either a peptide bond to a
further sequence of one or more amino acid residues or a covalent bond to
a carboxyl or hydroxyl end group.
Polypeptide and Peptide: Polypeptide and peptide are terms used
interchangeably herein to designate a linear series of amino acid residues
connected one to the other by peptide bonds between the alpha-amino and
carboxy groups of adjacent residues.
Protein: Protein is a term used herein to designate a linear series of
greater than about 50 amino acid residues connected one to the other as in
a polypeptide.
Synthetic peptide: refers to a chemically produced chain of amino acid
residues linked together by peptide bonds that is free of naturally
occurring proteins and fragments thereof.
Nucleotide: A monomeric unit of DNA or RNA consisting of a sugar moiety
(pentose), a phosphate, and a nitrogenous heterocyclic base. The base is
linked to the sugar moiety via the glycosidic carbon (1' of the pentose)
and that combination of base and sugar is a nucleoside. When the
nucleoside contains a phosphate group bonded to the 3' or 5' position of
the pentose it is referred to as a nucleotide. A sequence of operatively
linked nucleotides is typically referred to herein as a "base sequence" or
"nucleotide sequence", and their grammatical equivalents, and is
represented herein by a formula whose left to right orientation is in the
conventional direction of 5'-terminus to 3'-terminus.
Base Pair (bp): A partnership of adenine (A) with thymine (T), or of
cytosine (C) with guanine (G) in a double stranded DNA molecule. In RNA,
uracil (U) is substituted for thymine.
Nucleic Acid: A polymer of nucleotides, either single or double stranded.
Polynucleotide: a polymer of single or double stranded nucleotides. As used
herein "polynucleotide" and its grammatical equivalents will include the
full range of nucleic acids. A polynucleotide will typically refer to a
nucleic acid molecule comprised of a linear strand of two or more
deoxyribonucleotides and/or ribonucleotides. The exact size will depend on
many factors, which in turn depends on the ultimate conditions of use, as
is well known in the art. The polynucleotides of the present invention
include primers, probes, RNA/DNA segments, oligonucleotides or "oligos"
(relatively short polynucleotides), genes, vectors, plasmids, and the
like.
Gene: A nucleic acid whose nucleotide sequence codes for an RNA or
polypeptide. A gene can be either RNA or DNA.
Duplex DNA: a double-stranded nucleic acid molecule comprising two strands
of substantially complementary polynucleotides held together by one or
more hydrogen bonds between each of the complementary bases present in a
base pair of the duplex. Because the nucleotides that form a base pair can
be either a ribonucleotide base or a deoxyribonucleotide base, the phrase
"duplex DNA" refers to either a DNA-DNA duplex comprising two DNA strands
(ds DNA), or an RNA-DNA duplex comprising one DNA and one RNA strand.
Recombinant DNA (rDNA) molecule: a DNA molecule produced by operatively
linking two DNA segments. Thus, a recombinant DNA molecule is a hybrid DNA
molecule comprising at least two nucleotide sequences not normally found
together in nature. rDNA's not having a common biological origin, i.e.,
evolutionarily different, are said to be "heterologous".
Vector: a rDNA molecule capable of autonomous replication in a cell and to
which a DNA segment, e.g., gene or polynucleotide, can be operatively
linked so as to bring about replication of the attached segment. Vectors
capable of directing the expression of genes encoding for one or more
proteins are referred to herein as "expression vectors". Particularly
important vectors allow cloning of cDNA (complementary DNA) from mRNAs
produced using reverse transcriptase.
B. Integrin Alpha Subunit Polypeptides
The present invention relates to a previously undescribed species of
integrin alpha subunit that is derived by splicing of the messenger RNA in
the tissue in which the integrin alpha subunit is expressed, such that the
amino acid sequence of the alpha subunit polypeptide has a sequence as
defined herein.
Splicing as a form of regulation of gene expression is one means by which a
cell regulates the structural gene products expressed in that cell type.
According to the structures defined herein, it is now known that the
.alpha..sub.6 and .alpha..sub.3 integrin subunits can each be expressed in
two alternate forms (isoforms), designated herein as an "A" form and a "B"
form depending upon the spliced product, and are referred to as
.alpha..sub.6A or .alpha..sub.6B, and as .alpha..sub.3B or .alpha..sub.3B.
The newly described .alpha..sub.6B and .alpha..sub.3B subunits contain a
carboxyterminal amino acid residue sequence defining their cytoplasmic
domain that is different from their .alpha..sub.6A and .alpha..sub.3A
counterparts. These new species of .alpha..sub.6B and .alpha..sub.3B
provide, based on their sequence differences, novel polypeptide reagents
based on (1) the antigenic determinants present in their cytoplasmic
domains and (2) the structural role the cytoplasmic domain of these
proteins play in the function of the integrins of which they are members.
1. .alpha..sub.6B Subunit Polypeptides
In one embodiment, the present invention contemplates a polypeptide based
on the cytoplasmic domain of the .alpha..sub.6B species of the integrin
.alpha..sub.6 subunit. This polypeptide has an amino acid sequence that
includes a sequence that corresponds, and preferably is identical to, the
amino acid residue sequence of the cytoplasmic domain of the human or
mouse .alpha..sub.6B.
The cytoplasmic domain of human .alpha..sub.6B includes an amino acid
residue sequence shown in SEQ ID NO 3 from residue 1068 to residue 1091
and of mouse .alpha..sub.6B has an amino acid residue sequence shown in
SEQ ID NO 5 from residue 121 to residue 141.
Thus, in one embodiment, the present invention contemplates a polypeptide
having an amino acid residue sequence that includes at least the sequence
shown in SEQ ID NO 3 from residue 1068 to residue 1091 that defines the
carboxy terminal portion of cytoplasmic domain of human .alpha..sub.6B.
Preferably a polypeptide has an amino acid residue sequence shown in SEQ
ID NO 3 from residue 1068 to residue 1091, and more preferably has an
amino acid residue sequence shown in SEQ ID NO 3 from residue 1045 to
residue 1091. In a related embodiment the invention contemplates the whole
human .alpha..sub.6B protein, in a substantially isolated form, having a
sequence shown in SEQ ID NO 3 from residue 1 to residue 1091.
By substantially isolated is meant that the protein is present in a
composition as a major constituent, typically in amount greater than 10%,
and preferably greater than 90%, of the total protein in the composition.
Human .alpha..sub.6B protein can be isolated by a variety of biochemical
and immunological means from the tissue sources and cells described herein
that contain .alpha..sub.6B subunit. Exemplary methods involve the use of
a .alpha..sub.6B cytoplasmic domain specific antibody, such as 382
described herein, alone or in combination with the teachings of Kajiji et
al., EMBO J., 8:673-680 (1989).
In a related embodiment, the present invention contemplates a polypeptide
having an amino acid residue sequence that includes at least the sequence
shown in SEQ ID NO 5 from residue 121 to residue 141 that defines a
portion of the cytoplasmic domain of mouse .alpha..sub.6B. Preferably a
polypeptide has an amino acid residue sequence shown in SEQ ID NO 5 from
residue 121 to residue 141. Also contemplated is the whole mouse
.alpha..sub.6B protein in a substantially isolated form that included a
sequence shown in SEQ ID NO 5 from residue 1 to residue 141, with the
degree of isolation being the same as above for human .alpha..sub.6B.
Purification of mouse .alpha..sub.6B can similarly be accomplished using
the methods described above, and particularly using the murine cells
described herein as a source of protein and an anti-peptide antibody
prepared using mouse .alpha..sub.6B cytoplasmic domain-derived
polypeptides.
The native mouse .alpha..sub.6B subunit polypeptide is a protein of about
125,000 daltons in molecular weight when analysed by PAFE-SDS under
reducing conditons as described in the Examples.
The native human .alpha..sub.6B subunit polypeptide is a protein of about
125,000 daltons in molecular weight when analysed by polyacrylamide-sodium
dodecyl sulfate gel electrophoresis (PAGE-SDS) under reducing conditions
as described in the Examples.
2. .alpha..sub.3B Subunit Polypeptides
In another embodiment, the present invention contemplates a polypeptide
based on the cytoplasmic domain of the .alpha..sub.3B species of the
integrin .alpha..sub.3B subunit. This polypeptide has an amino acid
sequence that includes a sequence that corresponds, and preferably is
identical to, the amino acid residue sequence of the cytoplasmic domain of
the human or mouse .alpha..sub.3B.
The cytoplasmic domain of mouse .alpha..sub.3B has an amino acid residue
sequence shown in SEQ ID NO 9 from residue 113 to residue 153.
Thus, in one embodiment, the present invention contemplates a polypeptide
having an amino acid residue sequence that includes at least the sequence
shown in SEQ ID NO 9 from residue 113 to residue 153 that defines a
portion of the cytoplasmic domain of .alpha..sub.3B. Preferably a
polypeptide has an amino acid residue sequence shown in SEQ ID NO 9 from
residue 113 to residue 153, and more preferably has an amino acid residue
sequence shown in SEQ ID NO 9 from residue 1 to residue 153.
In a related embodiment, the invention contemplates the whole mouse
.alpha..sub.3B protein, in a substantially isolated form having a sequence
that includes the sequence shown in SEQ ID NO 9 from residue 1 to residue
153. The degree of isolation for mouse .alpha..sub.3B is the same as is
for human .alpha..sub.6B above, with methods for preparing the mouse
.alpha..sub.3B similarly based on immunoprecipitation or immunoaffinity
isolation methods using an antibody specific for mouse .alpha..sub.3B
cytoplasmic domain as defined herein.
In preferred embodiments, a polypeptide of the present invention comprises
about 20 to 1100 amino acid residues, and preferably comprises about 24 to
50 amino acid residues.
Preferably, a polypeptide of this invention is further characterized by its
ability to immunologically mimic an epitope (antigenic determinant)
expressed by the cytoplasmic domain of .alpha..sub.6B or .alpha..sub.3B as
defined herein.
As used herein, the phrase "immunologically mimic" in its various
grammatical forms refers to the ability of a polypeptide of this invention
to immunoreact with an antibody of the present invention that recognizes
an epitope on the cytoplasmic domain of .alpha..sub.6B or .alpha..sub.3B
as defined herein.
It should be understood that a subject polypeptide need not be identical to
the amino acid residue sequence of .alpha..sub.6B or .alpha..sub.3B so
long as it includes a sequence that provides at least one epitope within
the cytoplasmic domain of the .alpha..sub.6B or .alpha..sub.3B subunit and
is able to immunoreact with antibodies of the present invention.
A subject polypeptide includes any analog, fragment or chemical derivative
of a polypeptide whose amino acid residue sequence is shown herein so long
as the polypeptide is capable of immunologically mimicking a native
epitope present in the cytoplasmic domain of .alpha..sub.6B or
.alpha..sub.3B. Therefore, a polypeptide can be subject to various
changes, substitutions, insertions, and deletions where such changes
provide for certain advantages in its use. The term "analog" includes any
polypeptide having an amino acid residue sequence substantially identical
to a sequence specifically shown herein in which one or more residues have
been conservatively substituted with a functionally similar residue and
which displays the ability to mimic the cytoplasmic domain of
.alpha..sub.6B or .alpha..sub.3B as described herein. Examples of
conservative substitutions include the substitution of one non-polar
(hydrophobic) residue such as isoleucine, valine, leucine or methionine
for another, the substitution of one polar (hydrophilic) residue for
another such as between arginine and lysine, between glutamine and
asparagine, between glycine and serine, the substitution of one basic
residue such as lysine, arginine or histidine for another, or the
substitution of one acidic residue, such as aspartic acid or glutamic acid
for another.
The phrase "conservative substitution" also includes the use of a
chemically derivatized residue in place of a non-derivatized residue
provided that such polypeptide displays the requisite binding activity.
"Chemical derivative" refers to a subject polypeptide having one or more
residues chemically derivatized by reaction of a functional side group.
Such derivatized molecules include for example, those molecules in which
free amino groups have been derivatized to form amine hydrochlorides,
p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,
chloroacetyl groups or formyl groups. Free carboxyl groups may be
derivatized to form salts, methyl and ethyl esters or other types of
esters or hydrazides. Free hydroxyl groups may be derivatized to form
0-acyl or 0-alkyl derivatives. The imidazole nitrogen of histidine may be
derivatized to form N-im-benzylhistidine. Also included as chemical
derivatives are those peptides which contain one or more naturally
occurring amino acid derivatives of the twenty standard amino acids. For
examples: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine
may be substituted for lysine; 3-methylhistidine may be substituted for
histidine; homoserine may be substituted for serine; and ornithine may be
substituted for lysine. Polypeptides of the present invention also include
any polypeptide having one or more additions and/or deletions or residues
relative to the sequence of a polypeptide whose sequence is shown herein,
so long as the requisite activity is maintained.
The term "fragment" refers to any subject polypeptide having an amino acid
residue sequence shorter than that of a polypeptide whose amino acid
residue sequence is shown herein.
When a polypeptide of the present invention has a sequence that is not
identical to the sequence of the cytoplasmic domain of .alpha..sub.6B or
.alpha..sub.3B because one or more conservative or non-conservative
substitutions have been made, usually no more than about 30 number
percent, more usually no more than 20 number percent, and preferably no
more than 10 number percent of the amino acid residues are substituted,
except that additional residues can be added at either terminus for the
purpose of providing a "linker" by which the polypeptides of this
invention can be conveniently affixed to a label or solid matrix, or
carrier, such that the linker residues do not form epitopes expressed by
the cytoplasmic domain of .alpha..sub.6B or .alpha..sub.3B as defined
herein. Labels, solid matrices and carriers that can be used with the
polypeptides of this invention are described hereinbelow.
Amino acid residue linkers are usually at least one residue and can be 40
or more residues, more often 1 to 10 residues. Typical amino acid residues
used for linking are tyrosine, cysteine, lysine, glutamic and aspartic
acid, or the like. In addition, a subject polypeptide can differ, unless
otherwise specified, from the natural sequence of an .alpha..sub.6B or
.alpha..sub.3B cytoplasmic domain by the sequence being modified by
terminal-NH.sub.2 acylation, e.g., acetylation, or thioglycolic acid
amidation, by terminal-carboxlyamidation, e.g., with ammonia, methylamine,
and the like.
When coupled to a carrier to form what is known in the art as a
carrier-hapten conjugate, a polypeptide of the present invention is
capable of inducing antibodies that immunoreact with the cytoplasmic
domain of either human or mouse .alpha.6B or mouse .alpha..sub.3B. Where
the immunogen is an .alpha..sub.3B -derived polypeptide, the induced
antibodies immunoreact with the cytopolasmic domain of either human or
mouse .alpha..sub.3B. This cross-reactivity between human and mouse
cytoplasmic domains is shown by the disclosures herein. In view of the
well established principle of immunologic cross-reactivity, the present
invention therefore contemplates antigenically related variants of the
polypeptides of this invention. An "antigenically related variant" is a
subject polypeptide that is capable of inducing antibody molecules that
immunoreact with a subject polypeptide and with .alpha..sub.6B or
.alpha..sub.3B.
Any peptide of the present invention may be used in the form of a
pharmaceutically acceptable salt. Suitable acids which are capable of
forming salts with the peptides of the present invention include inorganic
acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric
acid, thiocyanic acid, sulfuric acid, phosphoric acetic acid, propionic
acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid,
succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid,
naphthalene sulfonic acid, sulfanilic acid or the like.
Suitable bases capable of forming salts with the peptides of the present
invention include inorganic bases such as sodium hydroxide, ammonium
hydroxide, potassium hydroxide and the like; and organic bases such as
mono-, di- and tri-alkyl and aryl amines (e.g. triethylamine, diisopropyl
amine, methyl amine, dimethyl amine and the like) and optionally
substituted ethanolamines (e.g. ethanolamine, diethanolamine and the
like).
A polypeptide of the present invention, also referred to herein as a
subject polypeptide, can be synthesized by any of the techniques that are
known to those skilled in the polypeptide art, including recombinant DNA
techniques. Synthetic chemistry techniques, such as a solid-phase
Merrifield-type synthesis, are preferred for reasons of purity, antigenic
specificity, freedom from undesired side products, ease of production and
the like. An excellent summary of the many techniques available can be
found in J. M. Steward and J. D. Young, "Solid Phase Peptide Synthesis",
W. H. Freeman Co., San Francisco, 1969; M. Bodanszky, et al., "Peptide
Synthesis", John Wiley & Sons, Second Edition, 1976 and J. Meienhofer,
"Hormonal Proteins and Peptides", Vol. 2, p. 46, Academic Press (New
York), 1983 for solid phase peptide synthesis, and E. Schroder and K.
Kubke, "The Peptides", Vol. 1, Academic Press (New York), 1965 for
classical solution synthesis, each of which is incorporated herein by
reference. Appropriate protective groups usable in such synthesis are
described in the above texts and in J. F. W. McOmie, "Protective Groups in
Organic Chemistry", Plenum Press, New York, 1973, which is incorporated
herein by reference.
In general, the solid-phase synthesis methods contemplated comprise the
sequential addition of one or more amino acid residues or suitably
protected amino acid residues to a growing peptide chain. Normally, either
the amino or carboxyl group of the first amino acid residue is protected
by a suitable, selectively removable protecting group. A different,
selectively removable protecting group is utilized for amino acids
containing a reactive side group such as lysine.
Using a solid phase synthesis as exemplary, the protected or derivatized
amino acid is attached to an inert solid support through its unprotected
carboxyl or amino group. The protecting group of the amino or carboxyl
group is then selectively removed and the next amino acid in the sequence
having the complimentary (amino or carboxyl) group suitably protected is
admixed and reacted under conditions suitable for forming the amide
linkage with the residue already attached to the solid support. The
protecting group of the amino or carboxyl group is then removed from this
newly added amino acid residue, and the next amino acid (suitably
protected) is then added, and so forth. After all the desired amino acids
have been linked in the proper sequence, any remaining terminal and side
group protecting groups (and solid support) are removed sequentially or
concurrently, to afford the final polypeptide.
An .alpha..sub.6B or .alpha..sub.3B -derived polypeptide can be used, inter
alia, in the diagnostic methods and systems of the present invention to
detect .alpha..sub.6B or .alpha..sub.3B present in a body sample, or can
be used to prepare an inoculum as described herein for the preparation of
antibodies that immunoreact with epitopes on the cytoplasmic domain of
either .alpha..sub.6B or .alpha..sub.3B.
C. DNA Segments
In living organisms, the amino acid residue sequence of a protein or
polypeptide is directly related via the genetic code to the
deoxyribonucleic acid (DNA) sequence of the structural gene that codes for
the protein. Thus, a structural gene can be defined in terms of the amino
acid residue sequence, i.e., protein or polypeptide, for which it codes.
An important and well known feature of the genetic code is its redundancy.
That is, for most of the amino acids used to make proteins, more than one
coding nucleotide triplet (codon) can code for or designate a particular
amino acid residue. Therefore, a number of different nucleotide sequences
may code for a particular amino acid residue sequence. Such nucleotide
sequences are considered functionally equivalent since they can result in
the production of the same amino acid residue sequence in all organisms.
Occasionally, a methylated variant of a purine or pyrimidine may be
incorporated into a given nucleotide sequence. However, such methylations
do not affect the coding relationship in any way.
In one embodiment the present invention contemplates an isolated DNA
segment that comprises a nucleotide base sequence that encodes a
polypeptide that includes the amino acid residue sequence defining the
cytoplasmic domain of .alpha..sub.6B or .alpha..sub.3B as defined herein.
A DNA segment therefor has a nucleotide sequence encoding the human or
mouse .alpha..sub.6B or mouse .alpha..sub.3B proteins, or at least
encoding the cytoplasmic domain of those proteins. The nucleotide
sequences are generally shown in SEQ ID NO 4 for human .alpha..sub.6B, NO
6 for mouse .alpha..sub.6B and NO 10 for mouse .alpha..sub.3B.
Preferred DNA segments include a nucleotide base sequence represented by
the base sequence contained in SEQ ID NO 4 from base 3279 to base 3418 and
defining a coding sequence that translates into the cytoplasmic domain of
.alpha..sub.6B. Particularly preferred is a nucleotide base sequence
represented by the sequence contained in SEQ ID NO 4 from base 147 to base
3418 that defines the .alpha..sub.6B integrin subunit. Corresponding
nucleotide sequences for mouse .alpha..sub.6B in SEQ ID NO 6 are also
contemplated.
In another embodiment, preferred DNA segments include a nucleotide base
sequence represented by the base sequence contained in SEQ ID NO 10 from
base 339 to base 463 and defining a coding sequence that translates into
the cytoplasmic domain of .alpha..sub.3B. Particularly preferred is a
nucleotide base sequence represented by the sequence contained in SEQ ID
NO 10 from base 1 to base 463 that defines the carboxy terminal portion of
the .alpha..sub.3B integrin subunit, including the cytoplasmic domain of
.alpha..sub.3B.
In preferred embodiments, the length of the nucleotide base sequence is no
more than about 3,000 bases, preferably no more than about 1,000 bases.
A purified DNA segment of this invention is substantially free of other
nucleic acids that do not contain the nucleotide base sequences specified
herein for a DNA segment of this invention, whether the DNA segment is
present in the form of a composition containing the purified DNA segment,
or as a solution suspension or particulate formulation. By substantially
free is means that the DNA segment is present as at least 10% of the total
nucleic acid present by weight, preferably greater than 50%, and more
preferably greater than 90% of the total nucleic acid by weight.
In preferred embodiments, a DNA segment of the present invention is bound
to a complementary DNA segment, thereby forming a double stranded DNA
segment. In addition, it should be noted that a double stranded DNA
segment of this invention preferably has a single stranded cohesive tail
at one or both of its termini.
A DNA segment of the present invention can easily be synthesized by
chemical techniques, for example, the phosphotriester method of Matteucci
et al., J. Am. Chem. Soc., 103:3185 (1981). (The disclosures of the art
cited herein are incorporated herein by reference.) Of course, by
chemically synthesizing the structural gene portion, any desired
modifications can be made simply by substituting the appropriate bases for
those encoding a native amino acid residue.
In addition, a DNA segment can be prepared by first synthesizing
oligonucleonucleotides that correspond to portions of the DNA segment,
which oligonucleotides are then assembled by hybridization and ligation
into a complete DNA segment. Such methods are also well known in the art.
See for example, Paterson et al., Cell, 48:441-452 (1987); and Lindley et
al., Proc.Natl. Acad. Sci., 85:9199-9203 (1988), where a recombinant
peptide, neutrophil-activated factor, was produced from the expression of
a chemically synthesized gene in E. coli.
A DNA expression vector of the present invention is a recombinant DNA
(rDNA) molecule adapted for receiving and expressing translatable DNA
sequences in the form of a fusion polypeptide of this invention. A DNA
expression vector is characterized as being capable of expressing, in a
compatible host, a structural gene product such as an .alpha..sub.6B or
.alpha..sub.3B polypeptide of the present invention.
As used herein, the term "vector" refers to a nucleic acid molecule capable
of transporting between different genetic environments another nucleic
acid to which it has been operatively linked. Preferred vectors are those
capable of autonomous replication and expression of structural gene
products present in the DNA segments to which they are operatively linked.
As used herein, the term "operatively linked", in reference to DNA
segments, describes that the nucleotide sequence is joined to the vector
so that the sequence is under the transcriptional and/or translation
control of the expression vector and can be expressed in a suitable host
cell.
The choice of vector to which a structural gene is operatively linked
depends directly, as is well known in the art, on the functional
properties desired, e.g., vector replication and protein expression, and
the host cell to be transformed, these being limitations inherent in the
art of constructing recombinant DNA molecules.
In preferred embodiments, the vector utilized includes a prokaryotic
replicon i.e., a DNA sequence having the ability to direct autonomous
replication and maintenance of the recombinant DNA molecule extra
chromosomally in a prokaryotic host cell, such as a bacterial host cell,
transformed therewith. Such replicons are well known in the art. In
addition, those embodiments that include a prokaryotic replicon also
include a gene whose expression confers a selective advantage, such as
drug resistance, to a bacterial host transformed therewith. Typical
bacterial drug resistance genes are those that confer resistance to
ampicillin or tetracycline. Vectors typically also contain convenient
restriction sites for insertion of translatable DNA sequences. Exemplary
vectors are the plasmids pUC8, pUC9, pBR322, and pBR329 available from
BioRad Laboratories, (Richmond, Calif.) and pPL and pKK223 available from
Pharmacia, (Piscataway, N.J.). Also contemplated are vctors for expressing
a DNA segment of this invention in a yeast or mammalian host cell.
DNA expression control sequences include both 5' and 3' elements, as is
well known, to form a cistron able to express a structural gene product.
The 5' control sequences define a promoter for initiating transcription
and a ribosome binding site operatively linked at the 5' terminus of the
upstream translatable DNA sequence.
To achieve high levels of gene expression in E. coli, it is necessary to
use not only strong promoters to generate large quantities of mRNA, but
also ribosome binding sites to ensure that the mRNA is efficiently
translated. In E. coli, the ribosome binding site includes an initiation
codon (AUG) and a sequence 3-9 nucleotides long located 3-11 nucleotides
upstream from the initiation codon [Shine et al., Nature, 254:34 (1975).
The sequence, AGGAGGU, which is called the Shine-Dalgarno (SD) sequence,
is complementary to the 3' end of E. coli 16S mRNA. Binding of the
ribosome to mRNA and the sequence at the 3' end of the mRNA can be
affected by several factors:
(i) The degree of complementarity between the SD sequence and 3' end of the
16S tRNA.
(ii) The spacing and possibly the DNA sequence lying between the SD
sequence and the AUG [Roberts et al., Proc. Natl. Acad. Sci. USA. 76:760
(1979a); Roberts et al., Proc. Natl. Acad. Sci. USA, 76:5596 (1979b);
Guarente et al., Science, 209:1428 (1980); and Guarente et al., Cell.
20:543 (1980).] Optimization is achieved by measuring the level of
expression of genes in plasmids in which this spacing is systematically
altered. Comparison of different mRNAs shows that there are statistically
preferred sequences from positions -20 to +13 (where the A of the AUG is
position 0) [Gold et al., Annu. Rev. Microbiol., 35:365 (1981)]. Leader
sequences have been shown to influence translation dramatically (Roberts
et al., 1979 a, b supra).
(iii) The nucleotide sequence following the AUG, which affects ribosome
binding [Taniguchi et al., J. Mol. Biol., 118:533 (1978)].
D. Antibodies and Monoclonal Antibodies
The term "antibody" in its various grammatical forms is used herein as a
collective noun that refers to a population of immunoglobulin molecules
and/or immunologically active portions of immunoglobulin molecules, i.e.,
molecules that contain an antibody combining site or paratope.
An "antibody combining site" is that structural portion of an antibody
molecule comprised of heavy and light chain variable and hypervariable
regions that specifically binds (immunoreacts with) antigen. The term
immunoreact in its various forms means specific binding between an
antigenic determinant-containing molecule and a molecule containing an
antibody combining site such as a whole antibody molecule or a portion
thereof.
The phrase "antibody molecule" in its various grammatical forms as used
herein contemplates both an intact immunoglobulin molecule and an
immunologically active portion of an immunoglobulin molecule.
Exemplary antibody molecules for use in the diagnostic methods and systems
of the present invention are intact immunoglobulin molecules,
substantially intact immunoglobulin molecules and those portions of an
immunoglobulin molecule that contain the paratope, including those
portions known in the art as Fab, Fab', F(ab').sub.2 and F(v).
Fab and F(ab').sub.2 portions of antibodies are prepared by the proteolytic
reaction of papain and pepsin, respectively, on substantially intact
antibodies by methods that are well known. See for example, U.S. Pat. No.
4,342,566 to Theofilopolous and Dixon. Fab' antibody portions are also
well known and are produced from F(ab').sub.2 portions followed by
reduction of the disulfide bonds linking the two heavy reduction of the
disulfide bonds linking the two heavy chain portions as with
mercaptoethanol, and followed by alkylation of the resulting protein
mercaptan with a reagent such as iodoacetamide. An antibody containing
intact antibody molecules are preferred, and are utilized as illustrative
herein.
An antibody of the present invention in one embodiment is an
anti-cytoplasmic .alpha..sub.6B domain antibody characterized as being
capable of immunoreacting with 1) human .alpha..sub.6B, and 2) a
polypeptide having a sequence shown in SEQ ID NO 3 from residue 1045 to
residue 1091.
In another embodiment an antibody of this invention is an anti-cytoplasmic
.alpha..sub.6B domain antibody characterized as being capable of
immunoreacting with 1) human .alpha..sub.6B, and 2) a polypeptide having a
sequence shown in SEQ ID NO 3 from residue 1068-1091.
In another emobdiment an antibody of this invention is an anti-cytoplasmic
.alpha..sub.6B domain antibody characterized as being capable of
immunoreacting with 1) mouse .alpha..sub.6B and 2) a polypeptide having a
sequence shown in SEQ ID NO 5 from residue 121 to residue 141.
In another embodiment, an anti-cytoplasmic .alpha..sub.3B domain antibody
is contemplated that is characterized as being capable of immunoreacting
with 1) mouse .alpha..sub.3B, and 2) the polypeptide having a sequence
shown in SEQ ID NO 9 from residue 113 to residue 153.
Antibody immunoreactivity with antigens containing a cytoplasmic domain as
defined above can be measured by a variety of immunological assays known
in the art. Exemplary immunoreaction of a subject antibody with
.alpha..sub.6B or .alpha..sub.3B polypeptides is described in Examples 2
and 4.
For example, immunoreaction with whole protein can be measured by the
immunoprecipitation procedures described in Example 2. Immunoreaction of
antibodies with polypeptides can be conveniently measured using ELISA as
described in U.S. Pat. Nos. 3,643,090; No. 3,850,752; or No. 4,016,043,
which are incorporated herein by reference, using the polypeptide in the
solid phase, as is well know.
An antibody of the present invention is typically produced by immunizing a
mammal with an inoculum containing a polypeptide of this invention and
thereby induce in the mammal antibody molecules having immunospecificity
for the polypeptide. Exemplary immunization procedures for preparing an
antibody of this invention are described in Example 2. The antibody
molecules are then collected from the mammal and isolated to the extent
desired by well known techniques such as, for example, by using DEAE
Sephadex to obtain the IgG fraction.
To enhance the specificity of the antibody, the antibodies may be purified
by immunoaffinity chromatography using solid phase-affixed immunizing
polypeptide. The antibody is contacted with the solid phase-affixed
immunizing polypeptide for a period of time sufficient for the polypeptide
to immunoreact with the antibody molecules to form a solid phase-affixed
immunocomplex. The bound antibodies are separated from the complex by
standard techniques.
The antibody so produced can be used, inter alia, in the diagnostic methods
and systems of the present invention to detect .alpha..sub.6B or
.alpha..sub.3B subunits present in a body sample. See, for example, the
methods described in Examples 2 and 4.
The word "inoculum" in its various grammatical forms is used herein to
describe a composition containing a polypeptide of this invention as an
active ingredient used for the preparation of antibodies against the
cytoplasmic domain of an .alpha..sub.6B or .alpha..sub.3B polypeptide.
When a polypeptide is used in an inoculum to induce antibodies it is to be
understood that the polypeptide can be used in various embodiments, e.g.,
alone or linked to a carrier as a conjugate, or as a polypeptide polymer.
However, for ease of expression and in context of a polypeptide inoculum,
the various embodiments of the polypeptides of this invention are
collectively referred to herein by the term "polypeptide", and its various
grammatical forms.
For a polypeptide that contains fewer than about 35 amino acid residues, it
is preferable to use the peptide bound to a carrier for the purpose of
inducing the production of antibodies.
One or more additional amino acid residues can be added to the amino- or
carboxy-termini of the polypeptide to assist in binding the polypeptide to
a carrier. Cysteine residues added at the amino- or carboxy-termini of the
polypeptide have been found to be particularly useful for forming
conjugates via disulfide bonds. However, other methods well known in the
art for preparing conjugates can also be used. Exemplary additional
linking procedures include the use of Michael addition reaction products,
dialdehydes such as glutaraldehyde, Klipstein, et al., J. Infect. Dis.,
147:318-326 (1983) and the like, or the use of carbodiimide technology as
in the use of a water-soluble carbodiimide to form amide links to the
carrier. For a review of protein conjugation or coupling through activated
functional groups, see Aurameas, et al., Scand. J. Immunol., 1:7-23
(1978).
Useful carriers are well known in the art, and are generally proteins
themselves. Exemplary of such carriers are keyhole limpet hemocyanin
(KLH), edestin, thyroglobulin, albumins such as bovine serum albumin (BSA)
or human serum albumin (HSA), red blood cells such as sheep erythrocytes
(SRBC), tetanus toxoid, cholera toxoid as well as polyamino acids such as
poly (D-lysine: D-glutamic acid), and the like.
The choice of carrier is more dependent upon the ultimate use of the
inoculum and is based upon criteria not particularly involved in the
present invention. For example, a carrier that does not generate an
untoward reaction in the particular animal to be inoculated should be
selected.
The present inoculum contains an effective, immunogenic amount of a
polypeptide of this invention, typically as a conjugate linked to a
carrier. The effective amount of polypeptide per unit dose sufficient to
induce an immune response to the immunizing polypeptide depends, among
other things, on the species of animal inoculated, the body weight of the
animal and the chosen inoculation regimen as is well known in the art.
Inocula typically contain polypeptide concentrations of about 10
micrograms to about 500 milligrams per inoculation (dose), preferably
about 50 micrograms to about 50 milligrams per dose.
The term "unit dose" as it pertains to the inocula refers to physically
discrete units suitable as unitary dosages for animals, each unit
containing a predetermined quantity of active material calculated to
produce the desired immunogenic effect in association with the required
diluent; i.e., carrier, or vehicle. The specifications for the novel unit
dose of an inoculum of this invention are dictated by and are directly
dependent on (a) the unique characteristics of the active material and the
particular immunologic effect to be achieved, and (b) the limitations
inherent in the art of compounding such active material for immunologic
use in animals, as disclosed in detail herein, these being features of the
present invention.
Inocula are typically prepared from the dried solid polypeptide-conjugate
by dispersing the polypeptide-conjugate in a physiologically tolerable
(acceptable) diluent such as water, saline or phosphate-buffered saline to
form an aqueous composition.
Inocula can also include an adjuvant as part of the diluent. Adjuvants such
as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA)
and alum are materials well known in the art, and are available
commercially from several sources.
The techniques of polypeptide conjugation or coupling through activated
functional groups presently known in the art are particularly applicable.
See, for example, Aurameas, et al., Scand. J. Immunol., Vol 8, Suppl.
7:7-23 (1978) and U.S. Pat. Nos. 4,493,795, No. 3,791,932 and No.
3,839,153. In addition, a site directed coupling reaction can be carried
out so that any loss of activity due to polypeptide orientation after
coupling can be minimized. See, for example, Rodwell et al., Biotech.,
3:889-894 (1985), and U.S. Pat. No. 4,671,958.
One or more additional amino acid residues may be added to the amino- or
carboxy-termini of the polypeptide to assist in binding the polypeptide to
form a conjugate. Cysteine residues, usually added at the carboxy-terminus
of the polypeptide, have been found to be particularly useful for forming
conjugates via disulfide bonds, but other methods well-known in the art
for preparing conjugates may be used.
A preferred antibody of this invention is a monoclonal antibody.
The phrase "monoclonal antibody" in its various grammatical forms refers to
a population of antibody molecules that contain only one species of
antibody combining site capable of immunoreacting with a particular
epitope. A monoclonal antibody thus typically displays a single binding
affinity for any epitope with which it immunoreacts. A monoclonal antibody
may therefore contain an antibody molecule having a plurality of antibody
combining sites, each immunospecific for a different epitope, e.g., a
bispecific monoclonal antibody.
A monoclonal antibody is typically composed of antibodies produced by
clones of a single cell called a hybridoma that secretes (produces) but
one kind of antibody molecule. The hybridoma cell is formed by fusing an
antibody-producing cell and a myeloma or other self-perpetuating cell
line. The preparation of such antibodies was first described by Kohler and
Milstein, Nature 256:495-497 (1975), which description is incorporated by
reference. The hybridoma supernates so prepared can be screened for the
presence of antibody molecules that immunoreact with a polypeptide of this
invention, or for inhibition of the natural function of an .alpha..sub.6B
or .alpha..sub.3B subunit.
Briefly, to form the hybridoma from which the monoclonal antibody
composition is produced, a myeloma or other self-perpetuating cell line is
fused with lymphocytes obtained from the spleen of a mammal hyperimmunized
with an antigen containing the cytoplasmic domain of .alpha..sub.6B or
.alpha..sub.3B, such as is present in a polypeptide of this invention. The
polypeptide-induced hybridoma technology is described by Niman et al.,
Proc. Natl. Sci., U.S.A., 80:4949-4953 (1983), which description is
incorporated herein by reference.
It is preferred that the myeloma cell line used to prepare a hybridoma be
from the same species as the lymphocytes. Typically, a mouse of the strain
129 GlX.sup.+ is the preferred mammal. Suitable mouse myelomas for use in
the present invention include the
hypoxanthine-aminopterin-thymidine-sensitive (HAT) cell lines
P3X63-Ag8.653, and Sp2/0-Ag14 that are available from the American Type
Culture Collection, Rockville, Md., under the designations CRL 1580 and
CRL 1581, respectively.
Splenocytes are typically fused with myeloma cells using polyethylene
glycol (PEG) 1500. Fused hybrids are selected by their sensitivity to HAT.
Hybridomas producing a monoclonal antibody of this invention are
identified using an immunoassay such as the immunoprecipitation protocol
described in Example 3.
A monoclonal antibody of the present invention can also be produced by
initiating a monoclonal hybridoma culture comprising a nutrient medium
containing a hybridoma that secretes antibody molecules of the appropriate
polypeptide specificity. The culture is maintained under conditions and
for a time period sufficient for the hybridoma to secrete the antibody
molecules into the medium. The antibody-containing medium is then
collected. The antibody molecules can then be further isolated by well
known techniques.
Media useful for the preparation of these compositions are both well known
in the art and commercially available and include synthetic culture media,
inbred mice and the like. An exemplary synthetic medium is Dulbecco's
minimal essential medium (DMEM; Dulbecco et al., Virol. 8:396 (1959))
supplemented with 4.5 gm/1 glucose, 20 mm glutamine, and 20% fetal calf
serum. An exemplary inbred mouse strain is the Balb/c.
The monoclonal antibodies of this invention can be used in the same manner
as disclosed herein for antibodies of the present invention.
For example, the monoclonal antibody can be used in the diagnostic methods
and systems disclosed herein where formation of a cytoplasmic
.alpha..sub.6B or .alpha..sub.3B domain-containing immunoreaction product
is desired.
Other methods of producing a monoclonal antibody, a hybridoma cell, or a
hybridoma cell culture are also well known. See, for example, the method
of isolating monoclonal antibodies from an immunological repertoire as
described by Sastry, et al., Proc. Natl. Acad. Sci., 86:5728-5732 (1989);
Huse et al., Science, 246:1275-1281 (1989); and Mullinax et al,
Proc.Natl.Acad.Sci.USA, 87:8095-8099 (1990).
Also contemplated by this invention is the hybridoma cell, and cultures
containing a hybridoma cell that produce a monoclonal antibody of this
invention.
D. Diagnostic Systems
The present invention also describes a diagnostic system, preferably in kit
form, for assaying for the presence of antigen having the cytoplasmic
domain of .alpha..sub.6B or .alpha..sub.3B in a body sample such as a
tissue, body fluid or the like body sample. A diagnostic system includes,
in an amount sufficient for at least one assay, a subject polypeptide
and/or a subject antibody or monoclonal antibody, as a separately packaged
immunochemical reagent. Instructions for use of the packaged reagent are
also typically included.
As used herein, the term "package" refers to a solid matrix or material
such as glass, plastic, paper, foil and the like capable of holding within
fixed limits a polypeptide, polyclonal antibody or monoclonal antibody of
the present invention. Thus, for example, a package can be a glass vial
used to contain milligram quantities of a contemplated polypeptide or it
can be a microtiter plate well to which microgram quantities of a
contemplated polypeptide have been operatively affixed, i.e., linked so as
to be capable of being immunologically bound by an antibody.
"Instructions for use" typically include a tangible expression describing
the reagent concentration or at least one assay method parameter such as
the relative amounts of reagent and sample to be admixed, maintenance time
periods for reagent/sample admixtures, temperature, buffer conditions and
the like.
In one embodiment, a diagnostic system for assaying for the presence of or
to quantitate .alpha..sub.6B or .alpha..sub.3B in a sample, such as blood,
plasma or serum, comprises a package containing at least one
.alpha..sub.6B or .alpha..sub.3B derived polypeptide of this invention
depending on whether .alpha..sub.6B or .alpha..sub.3B is to be detected,
respectively. In another embodiment, a diagnostic system of the present
invention for assaying for the presence or amount of .alpha..sub.6B or
.alpha..sub.3B in a sample further includes an antibody composition of
this invention. An exemplary diagnostic system is described in Example 4.
In preferred embodiments, a diagnostic system of the present invention
further includes a label or indicating means capable of signaling the
formation of an immunocomplex containing a polypeptide or antibody
molecule of the present invention.
The word "complex" as used herein refers to the product of a specific
binding reaction such as an antibody-antigen or receptor-ligand reaction.
Exemplary complexes are immunoreaction products.
As used herein, the terms "label" and "indicating means" in their various
grammatical forms refer to single atoms and molecules that are either
directly or indirectly involved in the production of a detectable signal
to indicate the presence of a complex. Any label or indicating means can
be linked to or incorporated in an expressed protein, polypeptide, or
antibody molecule that is part of an antibody or monoclonal antibody
composition of the present invention, or used separately, and those atoms
or molecules can be used alone or in conjunction with additional reagents.
Such labels are themselves well-known in clinical diagnostic chemistry and
constitute a part of this invention only insofar as they are utilized with
otherwise novel proteins methods and/or systems.
The labeling means can be a fluorescent labeling agent that chemically
binds to antibodies or antigens without denaturing them to form a
fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable
fluorescent labeling agents are fluorochromes such as fluorescein
isocyanate (FIC), fluorescein isothiocyante (FITC),
5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC),
tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200
sulphonyl chloride (RB 200 SC) and the like. A description of
immunofluorescence analysis techniques is found in DeLuca,
"Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis, et al.,
eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which is incorporated
herein by reference.
In preferred embodiments, the indicating group is an enzyme, such as
horseradish peroxidase (HRP), glucose oxidase, or the like. In such cases
where the principal indicating group is an enzyme such as HRP or glucose
oxidase, additional reagents are required to visualize the fact that a
receptor-ligand complex (immunoreactant) has formed. Such additional
reagents for HRP include hydrogen peroxide and an oxidation dye precursor
such as diaminobenzidine. An additional reagent useful with glucose
oxidase is 2,2'-azino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).
Radioactive elements are also useful labeling agents and are used
illustratively herein. An exemplary radiolabeling agent is a radioactive
element that produces gamma ray emissions. Elements which themselves emit
gamma rays, such as .sup.124 I, .sup.125 I, .sup.128 I, .sup.132 I and
.sup.51 Cr represent one class of gamma ray emissionproducing radioactive
element indicating groups. Particularly preferred is .sup.125 I. Another
group of useful labeling means are those elements such as .sup.11 C,
.sup.18 F, .sup.15 O and .sup.13 N which themselves emit positrons. The
positrons so emitted produce gamma rays upon encounters with electrons
present in the animal's body. Also useful is a beta emitter, such .sup.111
indium of .sup.3 H.
The linking of labels, i.e., labeling of, polypeptides and proteins is well
known in the art. For instance, antibody molecules produced by a hybridoma
can be labeled by metabolic incorporation of radioisotope-containing amino
acids provided as a component in the culture medium. See, for example,
Galfre et al., Meth. Enzymol., 73:3-46 (1981). The techniques of protein
conjugation or coupling through activated functional groups are
particularly applicable. See, for example, Aurameas, et al., Scand. J.
Immunol., Vol. 8 Suppl. 7:7-23 (1978), Rodwell et al., Biotech., 3:889-894
(1984), and U.S. Pat. No. 4,493,795.
The diagnostic systems can also include, preferably as a separate package,
a specific binding agent. A "specific binding agent" is a molecular entity
capable of selectively binding a reagent species of the present invention
or a complex containing such a species, but is not itself a polypeptide or
antibody molecule composition of the present invention. Exemplary specific
binding agents are second antibody molecules, complement proteins or
fragments thereof, S. aureus protein A, and the like. Preferably the
specific binding agent binds the reagent species when that species is
present as part of a complex.
In preferred embodiments, the specific binding agent is labeled. However,
when the diagnostic system includes a specific binding agent that is not
labeled, the agent is typically used as an amplifying means or reagent. In
these embodiments, the labeled specific binding agent is capable of
specifically binding the amplifying means when the amplifying means is
bound to a reagent species-containing complex.
The diagnostic kits of the present invention can be used in an "ELISA"
format to detect the quantity of .alpha..sub.6B or .alpha..sub.3B subunit
in a vascular fluid sample such as blood, serum, or plasma. "ELISA" refers
to an enzyme-linked immunosorbent assay that employs an antibody or
antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody
conjugate to detect and quantify the amount of an antigen present in a
sample. A description of the ELISA technique is found in Chapter 22 of the
4th Edition of Basic and Clinical Immunology by D. P. Sites et al.,
published by Lange Medical Publications of Los Altos, Calif. in 1982 and
in U.S. Pat. Nos. 3,654,090; No. 3,850,752; and No. 4,016,043, which are
all incorporated herein by reference.
Thus, in preferred embodiments, a polypeptide or an antibody of the present
invention can be affixed to a solid matrix to form a solid support that
comprises a package in the subject diagnostic systems.
A reagent is typically affixed to a solid matrix by adsorption from an
aqueous medium although other modes of affixation applicable to proteins
and polypeptides well known to those skilled in the art, can be used.
Useful solid matrices are also well known in the art. Such materials are
water insoluble and include the cross-linked dextran available under the
trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.);
agarose; beads of polystyrene beads about 1 micron to about 5 millimeters
in diameter available from Abbott Laboratories of North Chicago, Ill.;
polyvinyl chloride, polystyrene, cross-linked polyacrylamide,
nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or
tubes, plates or the wells of a microtiter plate such as those made from
polystyrene or polyvinylchloride.
The reagent species, labeled specific binding agent or amplifying reagent
of any diagnostic system described herein can be provided in solution, as
a liquid dispersion or as a substantially dry power, e.g., in lyophilized
form. Where the indicating means is an enzyme, the enzyme's substrate can
also be provided in a separate package of a system. A solid support such
as the before-described microtiter plate and one or more buffers can also
be included as separately packaged elements in this diagnostic assay
system.
The packaging materials discussed herein in relation to diagnostic systems
are those customarily utilized in diagnostic systems.
The term "package" refers to a solid matrix or material such as glass,
plastic (e.g., polyethylene, polypropylene and polycarbonate), paper, foil
and the like capable of holding within fixed limits a diagnostic reagent
such as a polypeptide, antibody or monoclonal antibody of the present
invention. Thus, for example, a package can be a bottle, vial, plastic and
plastic-foil laminated envelope or the like container used to contain a
contemplated diagnostic reagent or it can be a microtiter plate well to
which microgram quantities of a contemplated diagnostic reagent have been
operatively affixed, i.e., linked so as to be capable of being
immunologically bound by an antibody or polypeptide to be detected.
F. Assay Methods
The present invention contemplates various immunoassay methods for
determining the amount of .alpha..sub.6B or .alpha..sub.3B in a biological
sample using a polypeptide, polyclonal antibody or monoclonal antibody of
this invention as an immunochemical reagent to form an immunoreaction
product whose amount relates, either directly or indirectly, to the amount
of .alpha..sub.6B or .alpha..sub.3B in the sample.
Those skilled in the art will understand that there are numerous well known
clinical diagnostic chemistry procedures in which an immunochemical
reagent of this invention can be used to form an immunoreaction product
whose amount relates to the amount of .alpha..sub.6B or .alpha..sub.3B
present in a body sample. Thus, while exemplary assay methods are
described herein, the invention is not so limited.
Various heterogeneous and homogeneous protocols, either competitive or
noncompetitive, can be employed in performing an assay method of this
invention, including radioimmunoprecipitation (RIP), solid phase
immunoassay such as ELISA, in situ immunoreaction assays for direct
binding of antigen in tissue samples, and the like immunoassay protocols.
Generally, to detect the presence of an .alpha..sub.6B or .alpha..sub.3B
subunit or polypeptide in a patient, an aliquot (i.e., a predetermined
amount) of a body fluid sample, such as urine or a vascular fluid, namely
blood, plasma or serum from the patient, or a tissue sample prepared for
immunoreaction, is contacted by admixture (admixed), with an antibody
composition of the present invention to form an immunoreaction admixture.
The admixture is then maintained under biological assay conditions
(immunoreaction conditions) for a period of time sufficient for the
.alpha..sub.6B or .alpha..sub.3B antigen present in the sample to
immunoreact with (immunologically bind) a portion of the antibody
combining sites present in the antibody composition to form a
antigen-antibody molecule immunoreaction product (immunocomplex). The
complex can then be detected as described herein. The presence of the
complex is indicative of .alpha..sub.6B or .alpha..sub.3B subunit or
polypeptide in the sample.
Maintenance time periods sufficient for immunoreaction are well known and
are typically from about 10 minutes to about 16-20 hours at a temperature
of about 4.degree. C. to about 45.degree. C., with the time and
temperature typically being inversely related. For example, longer
maintenance times are utilized at lower temperatures, such as 16 hours at
4.degree. C., and shorter times for higher temperatures, such as 1 hour at
room temperature.
Biological assay conditions are those that maintain the biological activity
of the immunochemical reagents of this invention and the .alpha..sub.6B or
.alpha..sub.3B subunit or polypeptide sought to be assayed such that the
reagents retain their ability to form an immunoreaction product. Those
conditions include a temperature range of about 4.degree. C. to about
45.degree. C., a pH value of about 5 to about 9 and an ionic strength
varying from that of distilled water to that of about one molar sodium
chloride. Methods for optimizing such maintenance time periods and
biological assay conditions are well known in the art.
Determining the presence or amount of an .alpha..sub.6B or .alpha..sub.3B
containing immunoreaction product formed by the above maintenance step,
either directly or indirectly, can be accomplished by assay techniques
well known in the art, and typically depend on the type of indicating
means used.
In a direct binding assay format for detecting .alpha..sub.6B or
.alpha..sub.3B in a tissue sample such as a tissue section, the antibody
is reacted with the target antigen in situ to form the immunoreaction
complex. thereafter, the immunocomplex is detected thereby indicating the
presence of the antigen in the tissue. Exemplary and preferred in situ
immunoassay formats are described in Example 4. Alternatively, the direct
binding assay can be practiced with a fluid body sample believed to
contain .alpha..sub.6B or .alpha..sub.3B subunits or polypeptides.
Thus, in this embodiment, the direct assay comprises the steps of:
(a) admixing a tissue sample or body fluid sample with an antibody
composition of this invention immunospecific for a cytoplasmic domain of
either .alpha..sub.6B or .alpha..sub.3B as described herein to form an
immunoreaction admixture;
(b) maintaining said immunoreaction admixture under biological assay
conditions for a time period sufficient to form an immunoreaction product;
and
(c) detecting the presence, and preferably amount, of the immunoreaction
product formed phase in step (b), and thereby the amount of
presence/amount of .alpha..sub.6B or .alpha..sub.3B in the sample.
More preferably, detecting in step (c) is performed by the steps of:
(i) admixing the immunoreaction product formed in step (b) with an
indicating means to form a second reaction admixture;
(ii) maintaining the second reaction admixture for a time period sufficient
for said indication means to bind the immunoreaction product formed in
step (b) and form a second reaction product; and,
(iii) determining the presence and/or amount of indicating means in the
second reaction product, and thereby the presence of the immunoreaction
product formed in step (b). Particularly preferred is the use of a labeled
second antibody, immunospecific for the first antibody, as the indicating
means, and preferably the label is horseradish peroxidase. In one
embodiment, it is particularly preferred to use (1) mouse anti-cytoplasmic
domain .alpha..sub.6B polypeptide antibody in the antibody composition,
and (2) goat anti-mouse IgG antibodies labeled with horseradish peroxidase
as the indicating means.
In a preferred competition assay method, the immunoreaction admixture
described above further contains a solid phase having affixed thereto a
solid phase antigen comprising an .alpha..sub.6B or .alpha..sub.3B subunit
or polypeptide having an amino acid residue sequence that includes the
cytoplasmic domain of .alpha..sub.6B or .alpha..sub.3B, respectively, of
this invention. Thus, in this embodiment, the assay comprises the steps
of:
(a) admixing a body fluid sample with 1) an antibody composition of this
invention and 2) a solid support having affixed thereto (operatively
linked) an antigen comprising an .alpha..sub.6B or .alpha..sub.3B subunit
or polypeptide having an amino acid residue sequence that includes the
cytoplasmic domain of .alpha..sub.6B or .alpha..sub.3B of this invention,
or both, to form an immunoreaction admixture having both a liquid phase
and a solid phase;
(b) maintaining said immunoreaction admixture under biological assay
conditions for a time period sufficient to form an immunoreaction product
in the solid phase; and
(c) detecting the presence, and preferably amount, of the immunoreaction
product formed in the solid phase in step (b), and thereby the amount of
presence/amount of one or both of .alpha..sub.6B and .alpha..sub.3B in the
body fluid sample.
In another competition assay format the immunoreaction admixture contains
(1) a body fluid sample, preferably cell free, (2) an antibody of this
invention and (3) a labeled antigen comprising the cytoplasmic domain of
.alpha..sub.6B or .alpha..sub.3B, wherein the antibody is present in the
solid phase, being affixed to a solid support, to form a liquid and a
solid phase. In this embodiment, the admixed body fluid sample competes
with the labeled reagent for immunoreaction with the solid phase antibody
to form a solid phase immunoreaction product. Thereafter, the detection of
label in the solid phase correlates with the amount of .alpha..sub.6B or
.alpha..sub.3B in the admixed fluid sample.
In one embodiment, the detection of a polypeptide of this invention in a
body sample is utilized as a means to monitor the fate of therapeuticallly
administered .alpha..sub.6B or .alpha..sub.3B derived polypeptides
according to the therapeutic methods disclosed herein.
Also contemplated are immunological assays capable of detecting the
presence of immunoreaction product formation without the use of a label.
Such methods employ a "detection means", which means are themselves
well-known in clinical diagnostic chemistry and constitute a part of this
invention only insofar as they are utilized with otherwise novel
polypeptides, methods and systems. Exemplary detection means include
methods known as biosensors and include biosensing methods based on
detecting changes in the reflectivity of a surface, changes in the
absorption of an evanescent wave by optical fibers or changes in the
propagation of surface acoustical waves.
EXAMPLES
The following Examples illustrate, but do not limit, the present invention.
1. Polypeptides
Polypeptides were synthesized using the classical solid-phase technique
described by Merrifield, Adv. Enzymol., 32:221-96 (1969) as adapted for
use with a Model 430A automated peptide synthesizer (Applied Biosystems,
Foster City, Calif.). Polypeptide resins were cleaved by hydrogen
fluoride, extracted and analyzed for purity by high-performance liquid
chromatograph using a reverse-phase C18 column. (Waters Associates,
Milford, Mass.).
The amino acid residue sequence of the polypeptides and their designations
are as follows:
##STR1##
Polypeptide p.alpha..sub.6A has a sequence from the cytoplasmic domain of
.alpha..sub.6A and is shown in SEQ ID NO 1 from residue 1059 to residue
1073 to which an additional cysteine residue was included at the
N-terminus for coupling the peptide to a protein carrier (KLH) for
immunization. Polypeptide p.alpha..sub.6B has a sequence from the
cytoplasmic domain of .alpha..sub.6B and is shown in SEQ ID NO 3 from
residue 1068 to residue 1091 to which an additional cysteine residue was
included at the N-terminus for coupling the peptide to a protein carrier
(KLH) for immunization.
2. Preparation of Polyclonal Antisera
a. Conjugation of KLH
Briefly, as a generalized procedure for each polypeptide, 4 milligrams of
KLH in 0.25 milliliters (ml) of 10 millimolar (mM) sodium phosphate buffer
(pH 7.2) is reacted with 0.7 milligrams (mg) of MBS dissolved in DMF, and
the resulting admixture is stirred for 30 minutes at room temperature. The
MBS solution is added dropwise to ensure that the local concentration of
DMF was not too high, as KLH is insoluble at DMF concentrations of about
30% or higher. The reaction product, KLH-MB, is passed through a
chromatography column prepared with Sephadex G-25 (Pharmacia Fine
Chemicals, Piscataway, N.J.) equilibrated with 50 mM sodium phosphate
buffer (pH 6.0) to remove free MBS. KLH recovery from peak fractions of
the column eluate, monitored at 280 nanometers, is typically approximately
80%.
The KLH-MB so prepared is then reacted with 5 mg of polypeptide dissolved
in 1 ml of buffer. The pH value of the resulting reaction composition is
adjusted to 7-7.5, and the reaction composition is stirred at room
temperature for 3 hours to provide a polypeptide-carrier conjugate.
b. Immunization and Harvesting of Polyclonal Antisera
Inoculum stock solutions are prepared with CFA, IFA or alum as follows: An
amount of the synthetic polypeptide-conjugate sufficient to provide the
desired amount of polypeptide per inoculation is dissolved in
phosphate-buffered saline (PBS) at a pH value of 7.2. Equal volumes of
CFA, IFA or alum are then mixed with the polypeptide solution to provide
an inoculum containing polypeptide, water and adjuvant in which the
water-to-oil ratio is about 1:1. The mixture is thereafter homogenized to
provide the inoculum stock solution.
Rabbits used herein to raise anti-polypeptide antibodies were injected
subcutaneously with an inoculum comprising 200 micrograms (ug) of a
polypeptide conjugate (polypeptide plus carrier) emulsified in complete
Freund's adjuvant (CFA); 200 ug of polypeptide conjugate, incomplete in
Freund's adjuvant (IFA); and 200 ug of polypeptide conjugate with 4 mg
alum injected intraperitoneally on days 0, 14 and 21, respectively, of the
immunization schedule. Each inoculation (immunization) consisted of four
injections of the inoculum. Mice may be immunized in a similar way using
about one tenth of the above dose per injection.
Animals are typically bled 4 and 15 weeks after the first injection.
Control re-immune serum was obtained from each animal by bleeding just
before the initial immunization.
Control inoculum stock solutions can also be prepared with keyhole limpet
hemocyanin (KLH), KLH in IFA (incomplete Freund's adjuvant), KLH-alum
absorbed, KLH-alum absorbed-pertussis, edestin, thyroglobulin, tetanus
toxoid, tetanus toxoid in IFA, cholera toxoid and cholera toxoid in IFA.
Upon injection or other introduction of the antigen or inoculum into the
host animal, the immune system of the animal responds by producing large
amounts of antibody to the antigen. Since the specific antigenic
determinant of the manufactured antigen; i.e., the antigen formed form the
synthetic polypeptide linked to the carrier corresponds to the determinant
of the natural antigen of interest, the host animal manufactures
antibodies not only to the synthetic polypeptide to which the synthetic
polypeptide antigen corresponds; i.e., to the .alpha..sub.6B protein.
c. Immunoreactivity of Anti-peptide Antisera With Native .alpha..sub.6
Proteins
1. Protocols and Reagents
The rabbit polyclonal anti-.alpha..sub.6 cytoplasmic domain antiserum
designated 6844 was raised against the last 15 amino acids (SEQ ID NO 1,
residue 1059 to residue 1073) (IHAQPSDKERLTSDA) of the reported human
.alpha..sub.6 (.alpha..sub.6A) sequence (Tamura et al., J. Cell. Biol.,
111:1593-1604, 1990), to which an additional cystine residue was included
at the N-terminus for coupling the peptide to a protein carrier (KLH) for
immunization.
The rat monoclonal antibody, GoH3, is specific for an extracellular epitope
on both the human and murine .alpha..sub.6 subunits (Sonnenberg et al., J.
Biol. Chem., 262:10376-83, 1987). The isotype-matched control antibody,
B3B4, recognizes the B lymphocyte specific antigen, CD23.
The anti-.alpha..sub.6 specific monoclonal antibody, 35.13c, and the
isotype matched control antibody, 439.9b, specific for the human
.beta..sub.4 integrin subunit, have been previously described (Kennel et
al., J. Biol. Chem., 264:15515-21, 1989).
Anti-peptide antisera to the cytoplasmic domains of rat .alpha..sub.1,
chicken .alpha..sub.3, human .alpha..sub.4, human .alpha..sub.5 and human
.beta..sub.1 sequences were shown to be cross-reactive with the respective
were shown to be cross-reactive with the of B16F1 melanoma, STO fibroblast
and MMT carcinoma murine cell lines.
Antisera to the cytoplasmic domain of human .alpha..sub.6B were prepared by
immunizations of rabbits with the peptide p.alpha..sub.6B 1 having the
sequence (SEQ ID NO 3, residue 1068 to residue 1091 beginning at the
second residue of the following sequence) CDEKYIDNLEKKQWITKWNRNESYS as
described above to which an additional cysteine residue was included at
the N-terminus for coupling the peptide to a protein carrier (KLH) for
immunization. This antisera is designated 382.
The ES cells and B16Fl cell line were used in these immunoreaction studies.
The ES cell line, CCE (Schwartzberg et al., Science. 246:799-803, 1989)
was initially cultured on murine embryonic fibroblasts (STO cells) to
prevent differentiation. However, in order to study the expression and
function of integrins in this ES cell line it was necessary to remove the
STO cells from the culture system. Therefore, the CCE ES cell line was
subcloned into LIF (10.sup.3 units/ml) (Amrad Co. Australia) containing
media (DMEM; 10% FCS, 100mM .beta.-mercaptoethanol, 2mM glutamine). LIF
has been shown to prevent ES cell differentiation (Moreau et al.,Nature,
336:690-92, 1988; Smith et al.,Nature. 336:688-90, 1988; Williams et
al.,Nature, 336:684-687, 1988). The sublines were cultured on gelatin
(0.1%) coated plates. Several subclones were expanded and continually
cultured in LIF containing media. The subline ES1 was chosen for the
studies described here. ES1 cells were allowed to differentiate on gelatin
(0.1%) coated plates over a period of 8-9 days in the absence of LIF.
The murine B16FI melanoma line, obtained from Dr. Ralph Reisfeld (Scripps
Clinic, La Jolla, Calif.), was derived from a C57Bl/6 melanoma and
cultured in DMEM, 5% FCS, 2mM glutamine and penicillin-streptomycin (50
IU/ml-50ug/ml).
Undifferentiated ES cells (1-2.times.10.sup.7 cells) were surface labeled
with Na.sup.125 I using the lactoperoxidase procedure (Roth, Methods
Enzymol., 37(B):223-33, 1975). Differentiated ES cells proved to be
significantly more fragile than undifferentiated ES cells and did not
survive the more rigorous washing steps required during the iodination
procedure. Therefore, differentiated ES cells were metabolically labeled
with [.sup.35 S]methionine as described previously by Kajiji et al, EMBO
J., 8:673-680 (1989). Preparation of non-ionic detergent cell lysates,
immunoprecipitations and analysis by SDS-PAGE were performed as described
by Kajiji et al (1989), supra.
Immunoprecipitation is conducted generally by first admixing the rabbit
polyclonal antisera produced above with a cell lystate and maintaining the
admixture for a time period sufficient for immunocomplexes to form.
Thereafter, the immunoadsorbent Pansorbin (Sigma Chemical Co., St. Louis,
Mo.) is added to the admixture containing the innunocomplexes and
maintained to allow the Pansorbin to complex with (bind) the
immunocomplex. Thereafter the Pansorbin-containing bound immunocomplexes
are removed from the lysate admixture by centrifugation, washed several
times and the washed immunocomplexes are released from the Pansorbin and
analyzed by SDS-PAGE.
Sequential immunoprecipitation was also performed to identify the presence
of multiple immunoreactive species in a single lystate. After a first
immunoprecipitation as above the lysate is retained and subjected to a
second immunoprecipitation with unbound Pansorbin. The resulting lysate
from the second immunoprecipitation is again retained and subjected to a
third immunoprecipitation with unbound Pansorbin. Thus by the successive
rounds of sequential immunoprecipitation of a lysate using the same
antibody species, that lysate becomes depleted of antigen immunoreactive
with that antibody species. Thereafter, the depleted lysate is divided
into aliquots and each aliquot is separately immunoprecipitated (re-IP or
re-immunoprecipitated) using different antibodies. Antigens in the
depleteld lysate that immunoprecipitate with the second antibody different
from the depleting first antibody are not immunoreactive with the first
antibody. By sequential immunoprecipitation, two non-cross reacting
antigen species can be identified. As described herein, the cytoplasmic
domains of .alpha..sub.6A and .alpha..sub.6B are not cross reactive.
Mouse .alpha..sub.6
Separate immunoprecipitations were carried out on undifferentiated murine
ES1 cells with antiserum 382 raised against a synthetic peptide
corresponding to the sequence of the cytoplasmic tail of human
.alpha..sub.6B, with control preimmune serum from the same rabbit, and
with antisera 6844 directed to the cytoplasmic tail of human 6A. Only
antisera 382 precipitated protein bands virtually identical to those
reactive with anti-.alpha..sub.6 monoclonal GoH3 which is specific for
both .alpha..sub.6A and .alpha..sub.6B. These data indicated that ES1
cells do express .alpha..sub.6B protein, probably complexed with
.beta..sub.1, and antisera 382 is capable of recognizing the
.alpha..sub.6B protein.
In contrast to the immunoprecipitation data from undifferentiated ES1
cells, the anti-.alpha..sub.6A cytoplasmic domain polyclonal antiserum,
6844, could immunoprecipitate the .alpha..sub.6A isoform from .sup.35
S-methionine labelled lysates obtained from differentiated ES1 cells.
Thus, differentiation of ES1 cells is accompanied by the induction of
expression of the .alpha..sub.6A isoform.
The absence of the .alpha..sub.6A isoform in differentiated ES1 cells can
be seen by immunoprecipitations using GoH3 or 6844, which is shown in FIG.
1. Whereas the GoH3 antibody detects the lower molecular weight species
corresponding to .alpha..sub.6, the 6844 antibody, immunospecific for
.alpha..sub.6A, does not detect any .alpha..sub.6 species, indicating that
the GoH3-reactive form is an isoform, namely .alpha..sub.6B.
Similar immunoprecipitation assays were carried out on the D3 ES cell line
(see FIG. 1). The D3 embryonic stem cell line was derived by Doetschman et
al., J. Embryol. Exp. Morph., 87:27-45, (1985). D3 cells were cultured in
LIF containing medium as described above except that 15% FCS was used.
Immunoprecipitations of [.sup.35 S]methionine-labelled lysates showed that
the .alpha..sub.6B isoform is expressed at the protein level in both
undifferentiated and differentiated D3 cells while the .alpha..sub.6A
isoform was found only in the differentiated cells. This would suggest
that the ability to switch on .alpha..sub.6A expression upon
differentiation may be a general property of ES cells.
Because the 382 antisera was raised to a human .alpha..sub.6B cytoplasmic
domain-derived polypeptide and yet is shown above to immunoreact with the
mouse .alpha..sub.6B protein, the above data also shows that an
anti-.alpha..sub.6B antibody, whether raised to human or mouse varieties
of .alpha..sub.6B can be used to immunoreact with both human or mouse
.alpha..sub.6B.
Human .alpha..sub.6
Antisera 382 to a synthetic peptide corresponding to the last 25 residues
of human .alpha..sub.6B immunoprecipitated from radiolabeled detergent
lysates of the human choriocarcinoma cell line JAR (see Example 4 for
description of JAR cells) a pattern of bands similar or identical to those
obtained with 6844, an anti-peptide antiserum to the .alpha..sub.6A
cytoplasmic domain, and GoH3, a monoclonal antibody to the extracellular
domain of .alpha..sub.6 (see FIG. 2). The bands corresponded in molecular
weight to .alpha..sub.6, .beta..sub.1 and .beta..sub.4, and were
positively identified as such with specific antibodies. This result is
compatible with JAR cells expressing both .alpha..sub.6 .beta..sub.1 and
.alpha..sub.6 .beta..sub.4 heterodimers, and with PCR amplifications
detecting both .alpha..sub.6A and .alpha..sub.6B isoform bands in JAR
cells (see Example 3).
Sequential immunoprecipitations (FIG. 3) showed that antibody GoH3
completely depleted the JAR lysates of antigen reactive with antisera 382
(anti-.alpha..sub.6B) or 6844 (anti-.alpha..sub.6A). The 382 antiserum did
not remove any material reactive with 6844, and 6844 did not remove any
382-reactive material, while both antisera reduced, but did not completely
remove GoH3 reactivity (FIG. 2). These results indicate that JAR cells
express both .alpha..sub.6A and .alpha..sub.6B proteins, each of which is
paired with either .beta..sub.1 or .beta..sub.4. These results also
indicate that antisera raised to a mouse protein, namely the cytoplasmic
domain of mouse .alpha..sub.6B, immunoreacts with its human counterpart
protein, human .alpha..sub.6B.
3. Identification and Cloning of .alpha..sub.6B and .sub.3B cDNAs
cDNA molecules encoding human and mouse .alpha..sub.6B and mouse
.alpha..sub.3B cytoplasmic regions were prepared and fragments of the cDNA
molecules were selectively amplified using the polymerase chain reaction
(PCR) in the presence of specific oligonucleotide primers in order to
characterize gene expression of the .alpha..sub.6B and .alpha..sub.3B
proteins.
a. Procotols
Poly-A+mRNA was isolated from human JAR cells (Ameriacan Tissue Type
Collection, ATCC, Bethesda, Md., ATCC HTB 144), human U937 cells (ATCC CRL
1591), human FG cells (Dr. P. Meitner, Brown University) and both
differentiated and undifferentiated cell lines using the Invitrogen
Fastrack Kit (Invitrogen, La Jolla, Calif.). Single stranded cDNA was
synthesized from 10 ug of mRNA using AMV reverse transcriptase (20U;
Molecular Genetics Resources, Tampa, Fla.) and one ug of random hexamer
primers (Pharmacia). The cDNAs were extracted with phenol/chloroform, then
ethanol precipitated and about 0.5 to 10 ug cDNA was resuspended in
50-70ul of sterile water.
One ul of the resuspended cDNA was amplified per 50 ul PCR reaction mixture
(2.5mM MgC12, 50mM KCl, 10 mM .beta.-mercaptoethanol, 66 mM Tris.HCl;
pH8.3) using 0.1 uM oligonucleotide primers, 0.25 mM each of dATP, dTTP,
dCTP, and dGTP, and 1.25U of TAQ 1 polymerase (AmpliTaq; Perkin
Elmer/Cetus, Ca.). The PCR program consisted of 2 steps: (a) 40 cycles of
1 min at 94.degree. C, 2 min at 55.degree. C., and 3 min at 72.degree. C.
with a 5 sec/cycle extension on the 72.degree. C. segment, (b) 10 min at
72.degree. C. and a final shift to 4.degree. C. Second round PCR was
carried out on one ul of the reaction mixture generated from the first
round PCR.
Nested pairs of PCR primers were employed to ensure that .alpha..sub.6
specific fragments were amplified. Both sets of .alpha..sub.6 primers were
derived from the human .alpha..sub.6A cDNA sequence as determined by
Tamura et al., J. Cell. Biol., 111:1593-1604, (1990). The first set
corresponded to bp 2918-2937 (primer 1157) and 3454-3473 (primer 1156) of
the human .alpha..sub.6A sequence while the nested primer pair
corresponded to bp 2942-2960 (primer 1681) and 3433-3452 (primer 2002).
The sequence of these four primers are shown in SEQ ID NOS 11-14,
respectively. .alpha..sub.3 PCR primers designated primer 2032 and 2033
were derived from the hamster cDNA sequence as determined by Tsuji et al.,
J. Biol. Chem., 265:7016-7021 (1990). The sequence of primers 2032 and
2033 are shown in SEQ ID NOS 15 and 16, respectively.
Oligonucleotide primers were chemically synthesized by using a "Gene
Assembler" automated synthesizer (Pharmacia, Piscataway, N.J.).
Amplified fragments from first round PCR were purified using Gene Clean
(Bio 101, La Jolla, Calif.), treated with DNA polymerase I and T4
polynucleotide kinase, again purified with Gene Clean, and the blunt-ended
fragments were subcloned into the EcoRV site of
Bluescript-pKS+(Stratagene,La Jolla, Calif.). Clones containing insert
were sequenced manually (Sequenase kit; USB, Cleveland, Ohio) according to
the manufacturer's instructions using T3 and T7 polymerase vector primer
sequences. Sequences were analyzed on a VAX-VMS, version 5.2 computer,
with programs of the University of Wisconsin Genetics Computer Group
(Devereux et al, Nucl. Acids Res.,12:387-395, 1984).
b. Results
i. Human .alpha..sub.6
Oligonucleotides 1156 and 1157 flanking the 3' end of the coding region of
integrin .alpha..sub.6 mRNA were used as primers in polymerase chain
reactions (PCR). Two products, of 540 bp and 410 bp, were obtained using
first strand cDNAs from various cell lines as templates (FIG. 4). These
same products were obtained in second-round PCR with a nested set of
primers, indicating their specificity.
Both the 540bp and the 410bp PCR products were subcloned and sequenced. The
nucleotide sequence of the 540 bp fragment (designated .alpha..sub.6A in
FIG. 5) matches exactly the sequence of .alpha..sub.6A mRNA recently
reported by Tamura et al., J. Cell. Biol., 15 111:1593-1604, 1990, and
encodes the 3' portion of the end of the extracellular domain, the
transmembrane and the cytoplasmic domains, followed by the initial part of
the 3' untranslated region (3' UT).
The sequence of the 410 bp band matches the 540 bp sequence, with the
exception of a 130 bp gap shown in the lower sequence of FIG. 5, which
lower sequence corresponds to the nucleotide sequence of .alpha..sub.6B.
This gap corresponds to the region encoding the predicted .alpha..sub.6A
cytoplasmic domain, from the boundary with the transmembrane domain to 25
bp past the stop codon. Without this 130 bp segment, however, the open
reading frame continues in the previous 3' UT, resulting in an
.alpha..sub.6B protein with an alternative cytoplasmic domain (FIG. 6).
This alternative domain is 17 amino acid longer than, and bears no
sequence homology with, the reported .alpha.6A cytoplasmic domain, but it
does contain the sequence GFFKR, a motif present at the upstream border of
all mammalian integrin .alpha. chains sequenced. For convenience, the
published .alpha..sub.6 sequence is referred to as .alpha..sub.6A, and the
.alpha..sub.6 having the isoform cytoplasmic domain identified herein is
referred to as .alpha..sub.6B.
ii. Mouse .alpha..sub.6
Amplification of mouse .alpha..sub.6 cDNA expressed by undifferentiated ES1
and B16F1 cells was performed on first strand cDNA derived from these
mouse cells using the polymerase chain reaction (PCR). The nested sets of
PCR primers, pairs 1157/1156 and 1681/2002 described above, were employed.
FIG. 7A shows the PCR products obtained.
The PCR fragment amplified from B16Fl ("B16") cDNA correspond to the size
expected (510 bp) for the murine homologue of the human .alpha..sub.6
(FIG. 7A; lane 2). However, the PCR fragment obtained from the
amplification of the ES1 cell cDNA was significantly smaller.
Amplification of cDNAs derived from four independent ES1 mRNA preparations
yielded only the smaller fragment and never the larger fragment amplified
from B16F1 cDNA.
The PCR fragments from the ES1 and B16F1 cells were subcloned into the
Bluescript-pKS+vector and sequenced. FIG. 8 shows the nucleotide sequences
of the two PCR fragments. The sequence of the larger B16F1 fragment was
shown to be 89% identical to the human .alpha..sub.6A sequence at the
nucleotide level and 91% identical at the amino acid level, Tamura et al.,
J. Cell. Biol., 111:1593-1604 (1990). Thus the larger frament's sequence
represents the murine homologue of the human .alpha.6A subunit. The B16FI
PCR fragment (FIG. 8) encodes the C-terminal portion of the extracellular
domain as well as the transmembrane and cytoplasmic domains of the
.alpha..sub.6 subunit. Due to the selection of primers, additional coding
sequences 3' to the terminus of the sequence shown in FIG. 8 were not
detected. Thus, additional amino acid residues not shown in FIG. 8 are
present in the native mouse .alpha..sub.6B protein.
The sequence of the smaller PCR fragment (FIG. 8) was identical to the
B16F1 sequence except that an internal deletion of 130 bp was observed.
The location of the 130 bp deletion observed in the ES1 .alpha..sub.6 PCR
fragment exactly matched that of the human .alpha..sub.6B sequence.
Therefore, ES1 cells expressed the murine equivalent of the .alpha..sub.6B
isoform.
iii. Mouse .alpha..sub.3
Expression of the .alpha..sub.3 isoforms was also investigated in various
mouse tissues including muscle, heart, brain, lung and ovary. Using the
PCR procedure described above with the hamster .alpha..sub.3 primers, a
larger band corresponding to .alpha..sub.3A was amplified from most
tissues except heart, kidney, liver, thymus and spleen (Table 2;Example
4). A smaller band corresponding to .alpha..sub.3B was detected in heart
and brain. Cloning and sequencing of these bands showed that the larger
band corresponds exactly to the reported .alpha..sub.3 sequence
(.alpha..sub.3A), while the smaller band lacks a 144 bp segment and, like
.alpha..sub.6B, encodes an .alpha..sub.3 with an alternative cytoplasmic
tail (.alpha..sub.3B). The amino acid residue and nucleotide sequences of
the mouse .alpha..sub.3B cDNA-derived PCR fragments are shown in SEQ ID
NOS 9 and 10, respectively.
4. Tissue distribution of .alpha..sub.6A, .alpha..sub.6B, .alpha..sub.3A
and .alpha.hd 3B
a. PCR Amplification
The distribution of the .alpha..sub.6 isoforms in cultured cell lines and
mouse tissue was assessed by PCR as described in Example 3. The majority
of the cells tested contained both .alpha..sub.6A and .alpha..sub.6B mRNA
(see Tables 1 and 2). However, the two isoforms were reproducibly found at
ratios characteristic of a cell line. Interestingly, two carcinoma cell
lines and three lines of mouse embryonic fibroblasts (immortalized,
non-tranformed) contained exclusively .alpha..sub.6A, while embryonic stem
cells and F9 teratocarcinoma cells contained exclusively .alpha..sub.6B
(Table 1).
TABLE 1
______________________________________
CELL
LINE CELL TYPE .alpha..sub.6A
.alpha..sub.6B
.alpha..sub.3A
.alpha..sub.3B
______________________________________
FG Pancreatic Carcinoma
+ > + + -
1320 Met
" + > + ND ND
Panc-1 " + > + + -
SGR " + > + ND ND
JAR Choriocarcinoma
+ < + + -
JEG-3 " + < + ND ND
BeWo " + < + ND ND
LoVo Colon Carcinoma
+ < + ND ND
Colo 396
" + < + + -
CaCo-2 " + + + -
HT-29 " + > + + -
HeLa Cervical Carcinoma
+ > + ND ND
UCLA-P3 Lung Carcinoma + > + + -
A431 Epidermoid Carcinoma
+ - ND ND
K562 Erythroleukemia
+ + ND ND
U937 Histiocytic Lymphoma
+ > + + -
804G(Rat)
Bladder Carcinoma
+ - + -
3T3 (M) Embryonic Fibroblast
+ - + -
F9 (M) Teratocarcinoma
- + + -
ES (M) Embryonic Stem - + + -
ES (M) (Differentiated)
+ + ND ND
______________________________________
Cells were analysed by PCR amplification of .alpha..sub.6 and .alpha..sub.3
isoforms using the following human or mouse cells, with the cell sources
indicated in parenthesis: pancreatic carcinoma: FG, SGR and 1320 Met cells
(Dr. P. Meitner, Brown University); Panc-1 cells (ATCC CRL 1469);
choriocarcinoma: JAR cells (ATCC HTB 144); JEG-3 cells (ATCC HTB 36); BeWo
cells (ATCC CCL 98); colon carcinoma: LoVo cells (ATCC CCL 229 ); Colo 396
cells (Dr. T. Edgington, Scripps Clinic, La Jolla, Calif.); CaCo-2 cells
(ATCC HTB 37); HT-29 cells (ATCC HTB 38); Hela cervical carcinoma cells
(ATCC CCL 2); UCLA-P3 lung carcinoma cells (L. Walker, Scripps); A431
epidermoid carcinoma cells (ATCC CRL 1555); K562 erythroleukemia cells
(ATCC CCL 243); U937 histiocytic lymphoma cells (ATCC CCL 1593); 8049 rat
bladder carcinoma cells (J. Jones, Northwestern University, Evanston,
Ill.); NIH/3T3 mouse embryo fibroblasts (ATCC CRL 1658); F9 mouse
teratocarcinoma cells ATCC CRL 1720); ES mouse embryonic stem cells (E.
Robertson, Columbia University, N.Y.).
Table 1 illustrates the distinction of .alpha..sub.6A and .alpha..sub.6B,
and .alpha..sub.3A and .alpha..sub.3B subunit-encoding mRNAs in human and
mouse cultured cell lines. PCR amplification was performed on
single-stranded cDNA generated from each cell type, using oligonucleotides
specific for the .alpha..sub.6 or .alpha..sub.3 subunit, respectively. The
(+) symbol represents the presence of subunit-specific amplification
product in the tested sample, the (-) symbol represents its absence, and
(ND) indicates that analysis was not conducted on that tissue type. The
(>) is used when the .alpha..sub.6A subunit mRNA predominates over the
.alpha..sub.6B subunit mRNA, and the (<) symbol is used when the
.alpha..sub.6B subunit mRNA is the predominant species in the tissue.
By the same PCR assay, normal mouse lung, liver, spleen and cervix tissues
were solely .alpha..sub.6A, brain, ovary and kidney were solely
.alpha..sub.6B, while all other tissues tested contained both
.alpha..sub.6 isoforms (Table 2).
TABLE 2
______________________________________
TISSUE .alpha..sub.6A .alpha..sub.6B
.alpha..sub.3A
.alpha..sub.3B
______________________________________
Muscle + > + + -
Heart + > + - +
Kidney - + - -
Liver + - - -
Brain - + + < +
Lung + - + -
Stomach + < + ND ND
Intestine
+ < + ND ND
Cervix + - ND ND
Submax + + ND ND
Ovary - + + -
Thymus + > + - -
Spleen + - - -
______________________________________
Table 2 illustrates the distribution of .alpha..sub.6A and B and
.alpha..sub.3A and B subunit-encoding mRNAs in mouse tissues. The symbols
in Table 2 are the same as in Table 1.
Primary and nested PCR reactions were carried out on differentiated cell
lines as described in Example 3. ES1 cells were allowed to differentiate
over a period of 8-9 days in the absence of Leukemia Inhibitory Factor
(LIF). The morphology of the differentiated cells was dramatically
different from that of undifferentiated ES1 cells maintained in LIF. PCR
amplification on cDNA from undifferentiated ES1 cells, using .alpha..sub.6
specific primers, produced the 380 bp fragment corresponding to the
.alpha..sub.6B cytoplasmic sequence (FIG. 7B, lane 1). However, similar
amplification of cDNA from the differentiated cells produced two distinct
fragments of 510 bp and 380 bp (FIG. 7B, lane 2), shown by nucleotide
sequencing to be the .alpha..sub.6A and .alpha..sub.6B isoforms,
respectively.
b. In Situ Immunostaining of Tissues to Detect Tissue Distribution
Kidney biopsy materials were obtained by percutaneous needle biopsies using
modified Vim-Silverman needles in patients with glomerulonephritis. A
small portion of kidney biopsy materials were fixed with 4%
paraformaldehyde for 4 hours at 4.C and embedded in paraffin using an
automatic processor (Tissue-Tek.sup.R Rotary Tissue Processor). The tissue
was cut in 4 micron thickness with an AO rotary microtome, and
deparafinized with xylene or Histoclear (Baxter) and rehydrated with
graded alchohol. The rehydrated sections were washed with 0.1 M glycine in
TBS (0.005 M Tris-HCl; 0.9% NaCl, pH 7.5) for 5 minutes, treated with 0.1%
Triton X-100 for 2 min at room temperature (RT), and trypsinized (0.1%
trypsin for 5 min at 37.degree. C.). Nonspecific binding sites were
saturated by a blocking solution (5% dry milk solids, 1% heat inactivated
horse serum in TBS) for 30 minutes. Serially diluted primary antibodies [1
ug/ul of 33% saturated ammonium sulfate (SAS) cut of antisera 6844 and
382; 1:10 to 1:1000 dilutions in reagent diluent: i.e. 2.5% bovine serum
albumin in TBS] and normal control (SAS cut of normal rabbit serum,
lug/ul, 1:10 to 1:1000 dilution in reagent diluent) were incubated on
sections in humidified chambers at room temperature for 1 hr. Tissue
sections were further incubated with peroxidase conjugated goat
anti-rabbit antibody (Jackson Immunology) in reagent diluent (1:200) for
30 minutes. After this, 0.02% AEC (3-Amino-.alpha.-ethylcarbozole,
Aldrich) was applied for 30 minutes at room temperature. Each step was
followed by 3-minute washes in 0.005 M Tris-HCl, 0.9% NaCl pH 7.5 (TBS
wash). Washed tissue sections were counterstained with Mayor's hematoxylin
for 30 sec, mounted in Gel Mount (Biomeda) and observed under a light
microscope.
The results of the in situ immunostained kidney sections using the
anti-peptide antisera specific for .alpha..sub.6A (6844) or specific for
.alpha..sub.6B (382) are shown in FIG. 9. Panel A shows
anti-.alpha..sub.6A antibody molecules staining podocytes in the
glomelular structure of the
kidney but no staining in the tubules of the kidney. Panel B shows
anti-.alpha..sub.6B antibody molecules staining the epithelial cells of
the distal or collecting tubules of the kidney but not the glomerular
cells.
Additional kidney samples were similarly analysed and the results are shown
in Table 3.
TABLE 3.sup.1
______________________________________
Sera 6488 Sera 382
Patient G T G T
______________________________________
Normal - - - -
1 + - - +
2 ++ .+-. - ++
3 +++ - - +++
4 +++ - - ++++
5 ++++ - - ++++
______________________________________
.sup.1 "G" indicates the immunostaining pattern observed in glomerular
epithelial cells of the kidney, whereas "T" indicates the immunostaining
pattern observed in the tubular epithelial cells, where (-) indicates no
staining and + to ++++ indicates increasing intensities of stain. Patient
1-5 are patients that all have glomerular nephritis clinically indicated
as to require kidney biopsy.
The results in Table 3 indicate that in all patients exhibiting symptoms of
kidney dysfunction a distinct staining pattern is observed, namely that
antisera immunospecific for .alpha..sub.6A cytoplasmic domain (6488)
reacts with glomerular cells and antisera immunospecific for 2246B
cytoplasmic domain (382) reacts with the tubular epithelial cells.
These results show that antibodies immunoreactive with the cytoplasmic
domain of .alpha..sub.6B are useful for distinguishing cell types in
kidney sections, and particularly to identify distal and collecting
tubular epithelial cells in patients having conditions of kidney
dysfunction such as glomerular nephritis.
Although the present invention has now been described in terms of certain
preferred embodiments, and exemplified with respect thereto, one skilled
in the art will readily appreciate that various modifications, changes,
omissions and substitutions may be made without departing from the spirit
thereof. It is intended, therefore, that the present invention be limited
solely by the scope of the following claims.
SEQUENCE LISTINGS
SEQ ID NO 1 is the 1073 residue amino acid sequence of the human
.alpha..sub.6A protein. The putative transmembrane region is encompassed
by amino acids 1012-1037. The mature protein is cleaved from the signal
sequence between amino acids 23-24. Potential sites of N-linked
glycosylation are at positions 223, 284, 370, 513, 731, 748, 891, 927 and
958. Putative cation binding domains are at positions 230-238, 324-332,
386-394 and 441-449. The cytoplasmic sequence GFFKR, which is conserved in
virtually all of the integrin .alpha. chains, begins at amino acid
position 1040. The sequence encoded by the fragment of .alpha..sub.6A cDNA
amplified using primers 1156/1157 is encompassed by residues 927-1073.
##STR2##
SEQ ID NO 2 is the 5629 base nucleotide sequence of the human
.alpha..sub.6A cDNA. The initiating ATG is at nucleotide position 147. The
mature coding sequence begins at nucleotide position 216 and ends at
position 3365. The cytoplasmic sequence GFFKR is encoded by nucleotides
3264-3278. The 130 nucleotide sequence present in SEQ ID NO 2 but deleted
from SEQ ID NO 4 is encompassed by nucleotides 3261-3390. The sequence of
the .alpha..sub.6A cDNA amplified using primers 1156/1157 is encompassed
by nucleotides 2924-3455.
##STR3##
SEQ ID NO 3 is the 1091 residue amino acid sequence of the human
.alpha..sub.6B protein. The sequence of SEQ ID NO 3 is identical to SEQ ID
NO 1 between amino acids 1 and 1044. The sequence encoded by the fragment
of .alpha..sub.6B cDNA amplified using primers 1156/1157 is encompassed by
residue 927 through 1060.
##STR4##
SEQ ID NO 4 is the 5499 base nucleotide sequence of the human
.alpha..sub.6B cDNA. The sequence of SEQ ID NO 4 is identical to SEQ ID NO
2 between nucleotides 1 and 3260. Nucleotides 3261-5499 of SEQ ID NO 4 are
identical to nucleotides 3391-5629 of SEQ ID NO 2. SEQ ID NO 4 has a 130
nucleotide deletion in relation to SEQ ID NO 2. The sequence of the
.alpha..sub.6B cDNA amplified using primers 1156/1157 is encompassed by
nucleotides 2924-3325.
##STR5##
SEQ ID NO 5 is the 141 amino acid sequence predicted from the nucleic acid
product which results from amplification of the mouse .alpha..sub.6B cDNA
with primers 1157/1156. The putative transmembrane domain begins at amino
acid 88 and continues through amino acid 113. SEQ ID NO 5 is identical to
SEQ ID NO 7 at amino acid position 1 through 120; the two sequences
diverge at amino acid 121.
##STR6##
SEQ ID NO 6 is the 426 base nucleotide sequence corresponding to the mouse
.alpha..sub.6B amino acid sequence in SEQ ID NO 5. The putative
transmembrane region is encoded by nucleotides 262 through 337. SEQ ID NO
6 is identical to SEQ ID NO 8 except for 130 nucleotides present in SEQ ID
NO 8 but deleted between nucleotides 342 and 343 of SEQ ID NO 6.
##STR7##
SEQ ID NO 7 is the 149 amino acid sequence predicted from the product which
results from amplification of the mouse .alpha..sub.6A cDNA with primers
1157/1156. SEQ ID NO 7 is identical to SEQ ID NO 5 at amino acid positions
1 through 120; the sequences diverge at amino acid 121.
##STR8##
SEQ ID NO 8 is the 556 base nucleotide sequence corresponding to the mouse
.alpha..sub.6A amino acid sequence in SEQ ID NO 7, plus the first 109
nucleotides in the 3' noncoding region. SEQ ID NO 8 is identical to SEQ ID
NO 6 except it has a 130 base insertion (nucleotides 342-472 of SEQ ID NO
8) between nucleotides352 and 353 of SEQ ID NO 6.
##STR9##
SEQ ID NO 9 is the 153 amino acid sequence predicted from the product which
results from amplification of the mouse .alpha..sub.3B cDNA with primers
2032/2033. The cytoplasmic sequence CDFFK begins at amino acid position
108.
##STR10##
SEQ ID NO 10 is the 463 base nucleotide sequence corresponding to the mouse
.alpha..sub.3B amino acid sequence in SEQ ID NO 9. The cytoplasmic
sequence CDFFK is encoded by nucleotides 324-338.
##STR11##
SEQ ID NO 11 is the outer 5' PCR primer 1157, corresponding to bp 2918-2937
of the .alpha..sub.6A cDNA sequence of Sequence ID NO 2.
##STR12##
SEQ ID NO 12 is the outer 3' PCR primer 1156, corresponding to the
complement of bp 3454-3473 of the .alpha..sub.6A cDNA sequence of SEQ ID
NO 2.
##STR13##
SEQ ID NO 13 is the inner 5' nested PCR primer 1681, corresponding to bp
2942-2960 of the .alpha..sub.6A cDNA sequence of SEQ ID NO 2.
##STR14##
SEQ ID NO 14 is the inner 3' nested PCR primer 2002, corresponding to the
complement of bp 3433-3452 of the .alpha..sub.6A cDNA sequence of SEQ ID
NO 2.
##STR15##
SEQ ID NO 15 is the 5' PCR primer 2032, corresponding to the hamster
.alpha..sub.3A cDNA sequence of Tsuji et al., J. Biol. Chem.,
265:7016-7021 (1990).
##STR16##
SEQ ID NO 16 is the 3' PCR primer 2033, corresponding to the hamster
.alpha..sub.3A cDNA sequence of Tsuji et al., J. Biol. Chem.,
265:7016-7021 (1990).
##STR17##
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 16
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1073 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 1012..1037
(D) OTHER INFORMATION: /label=TRANSMEMBRANE
/note="The putative transmembrane region is
encompassed by amino acids 1012-1037."
(ix) FEATURE:
(A) NAME/KEY: Cleavage-site
(B) LOCATION: (23 24)
(D) OTHER INFORMATION: /note="The mature protein is
cleaved from the signal sequence between amino
acids 23- 24."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 223
(D) OTHER INFORMATION: /label=GLYCOSYLATION
/note="Potential site of N-linked glycosylation."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 284
(D) OTHER INFORMATION: /label=GLYCOSYLATION
/note="Potential site of N-linked glycosylation."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 370
(D) OTHER INFORMATION: /label=GLYCOSYLATION
/note="Potential site of N-linked glycosylation."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 513
(D) OTHER INFORMATION: /label=GLYCOSYLATION
/note="Potential site of N-linked glycosylation."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 731
(D) OTHER INFORMATION: /label=GLYCOSYLATION
/note="Potential site of N-linked glycosylation."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 748
(D) OTHER INFORMATION: /label=GLYCOSYLATION
/note="Potential site of N-linked glycosylation."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 891
(D) OTHER INFORMATION: /label=GLYCOSYLATION
/note="Potential site of N-linked glycosylation."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 927
(D) OTHER INFORMATION: /label=GLYCOSYLATION
/note="Potential site of N-linked glycosylation."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 958
(D) OTHER INFORMATION: /label=GLYCOSYLATION
/note="Potential site of N-linked glycosylation."
(ix) FEATURE:
(A) NAME/KEY: Binding-site
(B) LOCATION: 230..238
(D) OTHER INFORMATION: /note="Represents a putative
cation binding domain."
(ix) FEATURE:
(A) NAME/KEY: Binding-site
(B) LOCATION: 324..332
(D) OTHER INFORMATION: /note="Represents a putative
cation binding domain."
(ix) FEATURE:
(A) NAME/KEY: Binding-site
(B) LOCATION: 386..394
(D) OTHER INFORMATION: /note="Represents a putative
cation binding domain."
(ix) FEATURE:
(A) NAME/KEY: Binding-site
(B) LOCATION: 441..449
(D) OTHER INFORMATION: /note="Represents a putative
cation binding domain."
(ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 1040..1044
(D) OTHER INFORMATION: /label=CYTOPLASMIC
/note="The cytoplasmic sequence, which is
conserved in virtually all of the integrin ALPHA
chains."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: 927..1073
(D) OTHER INFORMATION: /note="The sequence encoded by the
fragment of ALPHA 6A cDNA amplified using primers
1156/1157."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
MetAlaAlaAlaGlyGlnLeuCysLeuLeuTyrLeuSerAlaGlyLeu
1510 15
LeuSerArgLeuGlyAlaAlaPheAsnLeuAspThrArgGluAspAsn
202530
ValIleArgLysTyrGlyAspProGlySerLeu PheGlyPheSerLeu
354045
AlaMetHisTrpGlnLeuGlnProGluAspLysArgLeuLeuLeuVal
505560
GlyAlaProArgGlyGluAlaLeuProLeuGlnArgAlaPheArgThr
65707580
GlyGlyLeuTyrSerCysAspIleThrAlaArgGlyPro CysThrArg
859095
IleGluPheAspAsnAspAlaAspProThrSerGluSerLysGluAsp
100105 110
GlnTrpMetGlyValThrValGlnSerGlnGlyProGlyGlyLysVal
115120125
ValThrCysAlaHisArgTyrGluLysArgGlnHisValAs nThrLys
130135140
GlnGluSerArgAspIlePheGlyArgCysTyrValLeuSerGlnAsn
145150155 160
LeuArgIleGluAspAspMetAspGlyGlyAspTrpSerPheCysAsp
165170175
GlyArgLeuArgGlyHisGluLysPheGlySerCysGln GlnGlyVal
180185190
AlaAlaThrPheThrLysAspPheHisTyrIleValPheGlyAlaPro
195200 205
GlyThrTyrAsnTrpLysGlyIleValArgValGluGlnLysAsnAsn
210215220
ThrPhePheAspMetAsnIlePheGluAspGlyProTyrGluValGly
225230235240
GlyGluThrGluHisAspGluSerLeuValProValProAlaAsnSer
245250 255
TyrLeuGlyPheSerLeuAspSerGlyLysGlyIleValSerLysAsp
260265270
GluIleThrPheValSerGlyAlaProArgAlaAsnHisS erGlyAla
275280285
ValValLeuLeuLysArgAspMetLysSerAlaHisLeuLeuProGlu
290295300
HisIlePheAspGlyGluGlyLeuAlaSerSerPheGlyTyrAspVal
305310315320
AlaValMetAspLeuAsnLysAspGlyTrpGlnAspIleValIl eGly
325330335
AlaProGlnTyrPheAspArgAspGlyGluValGlyGlyAlaValTyr
340345 350
ValTyrMetAsnGlnGlnGlyArgTrpAsnAsnValLysProIleArg
355360365
LeuAsnGlyThrLysAspSerMetPheGlyIleAlaValLysAsn Ile
370375380
GlyAspIleAsnGlnAspGlyTyrProAspIleAlaValGlyAlaPro
385390395400
TyrAspAspLeuGlyLysValPheIleTyrHisGlySerAlaAsnGly
405410415
IleAsnThrLysProThrGlnValLeuLysGlyIleSerPro TyrPhe
420425430
GlyTyrSerIleAlaGlyAsnMetAspLeuAspArgAsnSerTyrPro
435440445
AspValAlaValGlySerLeuSerAspSerValThrIlePheArgSer
450455460
ArgProValIleAsnIleGlnLysThrIleThrValThrProAsnArg
465470475480
IleAspLeuArgGlnLysThrAlaCysGlyAlaProSerGlyIleCys
48549049 5
LeuGlnValLysSerCysPheGluTyrThrAlaAsnProAlaGlyTyr
500505510
AsnProSerIleSerIleValGlyThrLeuGluAlaGluLysGl uArg
515520525
ArgLysSerGlyLeuSerSerArgValGlnPheArgAsnGlnGlySer
530535540
Glu ProLysTyrThrGlnGluLeuThrLeuLysArgGlnLysGlnLys
545550555560
ValCysMetGluGluThrLeuTrpLeuGlnAspAsnIleArgAspLys
565570575
LeuArgProIleProIleThrAlaSerValGluIleGlnGluProSer
580585590
SerArgArgArgValAsnSerLeuProGluValLeuProIleLeuAsn
595600605
SerAspGluProLysThrAlaHisIleAspValHisPheLeuLysGlu
610615620
GlyCysGlyAspAspAsnValCysAsnSerAsnLeuLysLeuGluTyr
625630635640
LysPheCysThrArgGluGlyAsnGlnAspLysPheSerTyrLeuPro
645650655
IleGlnLysGlyValProGluLeuValLeuLysAspGlnLysAspI le
660665670
AlaLeuGluIleThrValThrAsnSerProSerAsnProArgAsnPro
675680685
ThrLysAspGlyAspAspAlaHisGluAlaLysLeuIleAlaThrPhe
690695700
ProAspThrLeuThrTyrSerAlaTyrArgGluLeuArgAlaPhePro
705 710715720
GluLysGlnLeuSerCysValAlaAsnGlnAsnGlySerGlnAlaAsp
725730735
CysGluLeuGlyAsnProPheLysArgAsnSerAsnValThrPheTyr
740745750
LeuValLeuSerThrThrGluValThrPheAspThrProTyrLeuAsp
755760765
IleAsnLeuLysLeuGluThrThrSerAsnGlnAspAsnLeuAlaPro
770775780
IleThrA laLysAlaLysValValIleGluLeuLeuLeuSerValSer
785790795800
GlyValAlaLysProSerGlnValTyrPheGlyGlyThrValValGly
805810815
GluGlnAlaMetLysSerGluAspGluValGlySerLeuIleGluTyr
820825830
GluPheArgValIleAsnLeuGlyLysProLeuThrAsnLeuGlyThr
835840845
AlaThrLeuAsnIleGlnTrpProLysGluIleSerAsnGlyLysTrp
850855860
LeuLeuTyrLeuValLysValGluSerLysGlyLeuGluLysValThr
865870875880
Cys GluProGlnLysGluIleAsnSerLeuAsnLeuThrGluSerHis
885890895
AsnSerArgLysLysArgGluIleThrGluLysGlnIleAspAspAsn
900905910
ArgLysPheSerLeuPheAlaGluArgLysTyrGlnThrLeuAsnCys
915920925
Ser ValAsnValAsnCysValAsnIleArgCysProLeuArgGlyLeu
930935940
AspSerLysAlaSerLeuIleLeuArgSerArgLeuTrpAsnSerThr
945 950955960
PheLeuGluGluTyrSerLysLeuAsnTyrLeuAspIleLeuMetArg
965970975
A laPheIleAspValThrAlaAlaAlaGluAsnIleArgLeuProAsn
980985990
AlaGlyThrGlnValArgValThrValPheProSerLysThrValAla
99510001005
GlnTyrSerGlyValProTrpTrpIleIleLeuValAlaIleLeuAla
101010151020
GlyIleLeu MetLeuAlaLeuLeuValPheIleLeuTrpLysCysGly
1025103010351040
PhePheLysArgAsnLysLysAspHisTyrAspAlaThrTyrHisLys
104510501055
AlaGluIleHisAlaGlnProSerAspLysGluArgLeuThrSerAsp
106010651070
Ala
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5629 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 1..5629
(D) OTHER INFORMATION: /product="Human ALPHA 6A"
/note="SEQ ID NO: 2 is the 5629 base nucleotide
sequence of the human ALPHA 6A cDNA."
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 147..149
(D) OTHER INFORMATION: /function="Transcription
initiator"
(ix) FEATURE:
(A) NAME/KEY: matpeptide
(B) LOCATION: 216..3365
(D) OTHER INFORMATION: /product="Human ALPHA 6A"
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 3264..3278
(D) OTHER INFORMATION: /product="The cytoplasmic sequence
GFFKR."
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 3261..3390
(D) OTHER INFORMATION: /note= "The 130 nucleotide sequence
present in SEQ ID NO: 2 but deleted from SEQ ID
NO:4."
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 2924..3455
(D) OTHER INFORMATION: /note="The sequence of the ALPHA
6A cDNA amplified using primers 1156/1157."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GCGCGACCGTCCCGGGGGTGGGGCCGGGCGCA GCGGCGAGAGGAGGCGAAGGTGGCTGCG60
GTAGCAGCAGCGCGGCAGCCTCGGACCCAGCCCGGAGCGCAGGGCGGCCGCTGCAGGTCC120
CCGCTCCCCTCCCCGTGCGTCCGCCCATGGCCGCCGCCGGGCAGCTGTGCTTGCTCTACC180
TGTCGGCGGG GCTCCTGTCCCGGCTCGGCGCAGCCTTCAACTTGGACACTCGGGAGGACA240
ACGTGATCCGGAAATATGGAGACCCCGGGAGCCTCTTCGGCTTCTCGCTGGCCATGCACT300
GGCAACTGCAGCCCGAGGACAAGCGGCTGTTGCTCGTGGGGGCCCCGCGCGGAGA AGCGC360
TTCCACTGCAGAGAGCCTTCAGAACGGGAGGGCTGTACAGCTGCGACATCACCGCCCGGG420
GGCCATGCACGCGGATCGAGTTTGATAACGATGCTGACCCCACGTCAGAAAGCAAGGAAG480
ATCAGTGGATGGGGGTCACCGTCCAGAGCCAA GGTCCAGGGGGCAAGGTCGTGACATGTG540
CTCACCGATATGAAAAAAGGCAGCATGTTAATACGAAGCAGGAATCCCGAGACATCTTTG600
GGCGGTGTTATGTCCTGAGTCAGAATCTCAGGATTGAAGACGATATGGATGGGGGAGATT660
GGAGCTTTTG TGATGGGCGATTGAGAGGCCATGAGAAATTTGGCTCTTGCCAGCAAGGTG720
TAGCAGCTACTTTTACTAAAGACTTTCATTACATTGTATTTGGAGCCCCGGGTACTTATA780
ACTGGAAAGGGATTGTTCGTGTAGAGCAAAAGAATAACACTTTTTTTGACATGAA CATCT840
TTGAAGATGGGCCTTATGAAGTTGGTGGAGAGACTGAGCATGATGAAAGTCTCGTTCCTG900
TTCCTGCTAACAGTTACTTAGGTTTTTCTTTGGACTCAGGGAAAGGTATTGTTTCTAAAG960
ATGAGATCACTTTTGTATCTGGTGCTCCCAGA GCCAATCACAGTGGAGCCGTGGTTTTGC1020
TGAAGAGAGACATGAAGTCTGCACATCTCCTCCCTGAGCACATATTCGATGGAGAAGGTC1080
TGGCCTCTTCATTTGGCTATGATGTGGCGGTGATGGACCTCAACAAGGATGGGTGGCAAG1140
ATATAGTTAT TGGAGCCCCACAGTATTTTGATAGAGATGGAGAAGTTGGAGGTGCAGTGT1200
ATGTCTACATGAACCAGCAAGGCAGATGGAATAATGTGAAGCCAATTCGTCTTAATGGAA1260
CCAAAGATTCTATGTTTGGCATTGCAGTAAAAAATATTGGAGATATTAATCAAGA TGGCT1320
ACCCAGATATTGCAGTTGGAGCTCCGTATGATGACTTGGGAAAGGTTTTTATCTATCATG1380
GATCTGCAAATGGAATAAATACCAAACCAACACAGGTTCTCAAGGGTATATCACCTTATT1440
TTGGATATTCAATTGCTGGAAACATGGACCTT GATCGAAATTCCTACCCTGATGTTGCTG1500
TTGGTTCCCTCTCAGATTCAGTAACTATTTTCAGATCCCGGCCTGTGATTAATATTCAGA1560
AAACCATCACAGTAACTCCTAACAGAATTGACCTCCGCCAGAAAACAGCGTGTGGGGCGC1620
CTAGTGGGAT ATGCCTCCAGGTTAAATCCTGTTTTGAATATACTGCTAACCCCGCTGGTT1680
ATAATCCTTCAATATCAATTGTGGGCACACTTGAAGCTGAAAAAGAAAGAAGAAAATCTG1740
GGCTATCCTCAAGAGTTCAGTTTCGAAACCAAGGTTCTGAGCCCAAATATACTCA AGAAC1800
TAACTCTGAAGAGGCAGAAACAGAAAGTGTGCATGGAGGAAACCCTGTGGCTACAGGATA1860
ATATCAGAGATAAACTGCGTCCCATTCCCATAACTGCCTCAGTGGAGATCCAAGAGCCAA1920
GCTCTCGTAGGCGAGTGAATTCACTTCCAGAA GTTCTTCCAATTCTGAATTCAGATGAAC1980
CCAAGACAGCTCATATTGATGTTCACTTCTTAAAAGAGGGATGTGGAGACGACAATGTAT2040
GTAACAGCAACCTTAAACTAGAATATAAATTTTGCACCCGAGAAGGAAATCAAGACAAAT2100
TTTCTTATTT ACCAATTCAAAAAGGTGTACCAGAACTAGTTCTAAAAGATCAGAAGGATA2160
TTGCTTTAGAAATAACAGTGACAAACAGCCCTTCCAACCCAAGGAATCCCACAAAAGATG2220
GCGATGACGCCCATGAGGCTAAACTGATTGCAACGTTTCCAGACACTTTAACCTA TTCTG2280
CATATAGAGAACTGAGGGCTTTCCCTGAGAAACAGTTGAGTTGTGTTGCCAACCAGAATG2340
GCTCGCAAGCTGACTGTGAGCTCGGAAATCCTTTTAAAAGAAATTCAAATGTCACTTTTT2400
ATTTGGTTTTAAGTACAACTGAAGTCACCTTT GACACCCCATATCTGGATATTAATCTGA2460
AGTTAGAAACAACAAGCAATCAAGATAATTTGGCTCCAATTACAGCTAAAGCAAAAGTGG2520
TTATTGAACTGCTTTTATCGGTCTCGGGAGTTGCTAAACCTTCCCAGGTGTATTTTGGAG2580
GTACAGTTGT TGGCGAGCAAGCTATGAAATCTGAAGATGAAGTGGGAAGTTTAATAGAGT2640
ATGAATTCAGGGTAATAAACTTAGGTAAACCTCTTACAAACCTCGGCACAGCAACCTTGA2700
ACATTCAGTGGCCAAAAGAAATTAGCAATGGGAAATGGTTGCTTTATTTGGTGAA AGTAG2760
AATCCAAAGGATTGGAAAAGGTAACTTGTGAGCCACAAAAGGAGATAAACTCCCTGAACC2820
TAACGGAGTCTCACAACTCAAGAAAGAAACGGGAAATTACTGAAAAACAGATAGATGATA2880
ACAGAAAATTTTCTTTATTTGCTGAAAGAAAA TACCAGACTCTTAACTGTAGCGTGAACG2940
TGAACTGTGTGAACATCAGATGCCCGCTGCGGGGGCTGGACAGCAAGGCGTCTCTTATTT3000
TGCGCTCGAGGTTATGGAACAGCACATTTCTAGAGGAATATTCCAAACTGAACTACTTGG3060
ACATTCTCAT GCGAGCCTTCATTGATGTGACTGCTGCTGCCGAAAATATCAGGCTGCCAA3120
ATGCAGGCACTCAGGTTCGAGTGACTGTGTTTCCCTCAAAGACTGTAGCTCAGTATTCGG3180
GAGTACCTTGGTGGATCATCCTAGTGGCTATTCTCGCTGGGATCTTGATGCTTGC TTTAT3240
TAGTGTTTATACTATGGAAGTGTGGTTTCTTCAAGAGAAATAAGAAAGATCATTATGATG3300
CCACATATCACAAGGCTGAGATCCATGCTCAGCCATCTGATAAAGAGAGGCTTACTTCTG3360
ATGCATAGTATTGATCTACTTCTGTAATTGTG TGGATTCTTTAAACGCTCTAGGTACGAT3420
GACAGTGTTCCCCGATACCATGCTGTAAGGATCCGGAAAGAAGAGCGAGAGATCAAAGAT3480
GAAAAGTATATTGATAACCTTGAAAAAAAACAGTGGATCACAAAGTGGAACAGAAATGAA3540
AGCTACTCAT AGCGGGGGCCTAAAAAAAAAAAAGCTTCACAGTACCCAAACTGCTTTTTC3600
CAACTCAGAAATTCAATTTGGATTTAAAAGCCTGCTCAATCCCTGAGGACTGATTTCAGA3660
GTGACTACACACAGTACGAACCTACAGTTTTAACTGTGGATATTGTTACGTAGCC TAAGG3720
CTCCTGTTTTGCACAGCCAAATTTAAAACTGTTGGAATGGATTTTTCTTTAACTGCCGTA3780
ATTTAACTTTCTGGGTTGCCTTTGTTTTTGGCGTGGCTGACTTACATCATGTGTTGGGGA3840
AGGGCCTGCCCAGTTGCACTCAGGTGACATCC TCCAGATAGTGTAGCTGAGGAGGCACCT3900
ACACTCACCTGCACTAACAGAGTGGCCGTCCTAACCTCGGGCCTGCTGCGCAGACGTCCA3960
TCACGTTAGCTGTCCCACATCACAAGACTATGCCATTGGGGTAGTTGTGTTTCAACGGAA4020
AGTGCTGTCT TAAACTAAATGTGCAATAGAAGGTGATGTTGCCATCCTACCGTCTTTTCC4080
TGTTTCCTAGCTGTGTGAATACCTGCTCACGTCAAATGCATACAAGTTTCATTCTCCCTT4140
TCACTAAAAACACACAGGTGCAACAGACTTGAATGCTAGTTATACTTATTTGTAT ATGGT4200
ATTTATTTTTTCTTTTCTTTACAAACCATTTTGTTATTGACTAACAGGCCAAAGAGTCTC4260
CAGTTTACCCTTCAGGTTGGTTTAATCAATCAGAATTAGAATTAGAGCATGGGAGGGTCA4320
TCACTATGACCTAAATTATTTACTGCAAAAAG AAAATCTTTATAAATGTACCAGAGAGAG4380
TTGTTTTAATAACTTATCTATAAACTATAACCTCTCCTTCATGACAGCCTCCACCCCACA4440
ACCCAAAAGGTTTAAGAAATAGAATTATAACTGTAAAGATGTTTATTTCAGGCATTGGAT4500
ATTTTTTACT TTAGAAGCCTGCATAATGTTTCTGGATTTACATACTGTAACATTCAGGAA4560
TTCTTGGAGAAGATGGGTTTATTCACTGAACTCTAGTGCGGTTTACTCACTGCTGCAAAT4620
ACTGTATATTCAGGACTTGAAAGAAATGGTGAATGCCTATGGAACTAGTGGATCC AAACT4680
GATCCAGTATAAGACTACTGAATCTGCTACCAAAACAGTTAATCAGTGAGTCGAGTGTTC4740
TATTTTTTGTTTTGTTTCCTCCCCTATCTGTATTCCCAAAAATTACTTTGGGGCTAATTT4800
AACAAGAACTTTAAATTGTGTTTTAATTGTAA AAATGGCAGGGGGTGGAATTATTACTCT4860
ATACATTCAACAGAGACTGAATAGATATGAAAGCTGATTTTTTTTAATTACCATGCTTCA4920
CAATGTTAAGTTATATGGGGAGCAACAGCAAACAGGTGCTAATTTGTTTTGGATATAGTA4980
TAAGCAGTGT CTGTGTTTTGAAAGAATAGAACACAGTTTGTAGTGCCACTGTTGTTTTGG5040
GGGGGGCTTTTTTTCTTTTTCCGGAAAATCCTTAAACCTTAAGATACTAAGGACGTTGTT5100
TTGGTTGTACTTGGAATTCTTAGTCACAAAATATATTTTGTTTACAAAAATTTCT GTAAA5160
ACAGGTTATAACAGTGTTTAAAGTCTCAGTTTCTTGCTTGGGGAACTTGTGTCCCTAATG5220
TGTTAGATTGCTAGATTGCTAAGGAGCTGATACTTGACAGTTTTTTAGACCTGTGTTACT5280
AAAAAAAAGATGAATGTCGGAAAAGGGTGTTG GGAGGGTGGTCAACAAAGAAACAAAGAT5340
GTTATGGTGTTTAGACTTATGGTTGTTAAAAATGTCATCTCAAGTCAAGTCACTGGTCTG5400
TTTGCATTTGATACATTTTTGTACTAACTAGCATTGTAAAATTATTTCATGATTAGAAAT5460
TACCTGTGGA TATTTGTATAAAAGTGTGAAATAAATTTTTTATAAAAGTGTTCATTGTTT5520
CGTAACACAGCATTGTATATGTGAAGCAAACTCTAAAATTATAAATGACAACCTGAATTA5580
TCTATTTCATCAAAAAAAAAAAAAAAAAAAACTTTATGGGCACAACTGG 5629
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1091 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: 1..1091
(D) OTHER INFORMATION: /note="SEQ ID NO:3 is the 1091
residue amino acid sequence of the human ALPHA 6B
protein."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: 1..1044
(D) OTHER INFORMATION: /note="The sequence of SEQ ID NO:3
is identical to SEQ ID NO:1 between amino acids 1
and 1044."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: 927..1060
(D) OTHER INFORMATION: /note="Encompasses the sequence
encoded by the fragment of ALPHA 6B cDNA amplified
using primers 1156/1157."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
MetAlaAlaAlaGlyGlnLeuCysLeuLeuTyrLeuSerAlaGlyLeu
151015
LeuSerArgLeuGlyAlaAlaPheAsnLeuAspThrArgGluAspAsn
202530
ValIleArgLysTyrGlyAspProGlySerLeuPheGlyPheSerLeu
354045
AlaMetHisTrpGlnLeuGlnProGluAspLysArgLeuLeuLeuVal
505560
GlyAlaProArgGlyGluAlaLeuProLeuGlnArgAlaPheArgThr
65707580
GlyG lyLeuTyrSerCysAspIleThrAlaArgGlyProCysThrArg
859095
IleGluPheAspAsnAspAlaAspProThrSerGluSerLysGluAsp
100105110
GlnTrpMetGlyValThrValGlnSerGlnGlyProGlyGlyLysVal
115120125
ValThr CysAlaHisArgTyrGluLysArgGlnHisValAsnThrLys
130135140
GlnGluSerArgAspIlePheGlyArgCysTyrValLeuSerGlnAsn
145 150155160
LeuArgIleGluAspAspMetAspGlyGlyAspTrpSerPheCysAsp
165170175
Gly ArgLeuArgGlyHisGluLysPheGlySerCysGlnGlnGlyVal
180185190
AlaAlaThrPheThrLysAspPheHisTyrIleValPheGlyAlaPro
195200205
GlyThrTyrAsnTrpLysGlyIleValArgValGluGlnLysAsnAsn
210215220
ThrPhePheAspM etAsnIlePheGluAspGlyProTyrGluValGly
225230235240
GlyGluThrGluHisAspGluSerLeuValProValProAlaAsnSer
245250255
TyrLeuGlyPheSerLeuAspSerGlyLysGlyIleValSerLysAsp
260265270
GluIl eThrPheValSerGlyAlaProArgAlaAsnHisSerGlyAla
275280285
ValValLeuLeuLysArgAspMetLysSerAlaHisLeuLeuProGlu
290 295300
HisIlePheAspGlyGluGlyLeuAlaSerSerPheGlyTyrAspVal
305310315320
AlaValMet AspLeuAsnLysAspGlyTrpGlnAspIleValIleGly
325330335
AlaProGlnTyrPheAspArgAspGlyGluValGlyGlyAlaValTyr
340345350
ValTyrMetAsnGlnGlnGlyArgTrpAsnAsnValLysProIleArg
355360365
LeuAsnGly ThrLysAspSerMetPheGlyIleAlaValLysAsnIle
370375380
GlyAspIleAsnGlnAspGlyTyrProAspIleAlaValGlyAlaPro
385 390395400
TyrAspAspLeuGlyLysValPheIleTyrHisGlySerAlaAsnGly
405410415
IleAsnT hrLysProThrGlnValLeuLysGlyIleSerProTyrPhe
420425430
GlyTyrSerIleAlaGlyAsnMetAspLeuAspArgAsnSerTyrPro
435440445
AspValAlaValGlySerLeuSerAspSerValThrIlePheArgSer
450455460
ArgProValIleAsnIl eGlnLysThrIleThrValThrProAsnArg
465470475480
IleAspLeuArgGlnLysThrAlaCysGlyAlaProSerGlyIleCys
485490495
LeuGlnValLysSerCysPheGluTyrThrAlaAsnProAlaGlyTyr
500505510
AsnProSer IleSerIleValGlyThrLeuGluAlaGluLysGluArg
515520525
ArgLysSerGlyLeuSerSerArgValGlnPheArgAsnGlnGlySer
530 535540
GluProLysTyrThrGlnGluLeuThrLeuLysArgGlnLysGlnLys
545550555560
ValCysMetGlu GluThrLeuTrpLeuGlnAspAsnIleArgAspLys
565570575
LeuArgProIleProIleThrAlaSerValGluIleGlnGluProSer
580585590
SerArgArgArgValAsnSerLeuProGluValLeuProIleLeuAsn
595600605
SerAspGluProL ysThrAlaHisIleAspValHisPheLeuLysGlu
610615620
GlyCysGlyAspAspAsnValCysAsnSerAsnLeuLysLeuGluTyr
62563 0635640
LysPheCysThrArgGluGlyAsnGlnAspLysPheSerTyrLeuPro
645650655
IleGlnLysGl yValProGluLeuValLeuLysAspGlnLysAspIle
660665670
AlaLeuGluIleThrValThrAsnSerProSerAsnProArgAsnPro
675 680685
ThrLysAspGlyAspAspAlaHisGluAlaLysLeuIleAlaThrPhe
690695700
ProAspThrLeuThrTyrSer AlaTyrArgGluLeuArgAlaPhePro
705710715720
GluLysGlnLeuSerCysValAlaAsnGlnAsnGlySerGlnAlaAsp
725730735
CysGluLeuGlyAsnProPheLysArgAsnSerAsnValThrPheTyr
740745750
LeuValLeuSer ThrThrGluValThrPheAspThrProTyrLeuAsp
755760765
IleAsnLeuLysLeuGluThrThrSerAsnGlnAspAsnLeuAlaPro
770 775780
IleThrAlaLysAlaLysValValIleGluLeuLeuLeuSerValSer
785790795800
GlyValAlaLysProS erGlnValTyrPheGlyGlyThrValValGly
805810815
GluGlnAlaMetLysSerGluAspGluValGlySerLeuIleGluTyr
82 0825830
GluPheArgValIleAsnLeuGlyLysProLeuThrAsnLeuGlyThr
835840845
AlaThrLeuAsnIleGl nTrpProLysGluIleSerAsnGlyLysTrp
850855860
LeuLeuTyrLeuValLysValGluSerLysGlyLeuGluLysValThr
865870 875880
CysGluProGlnLysGluIleAsnSerLeuAsnLeuThrGluSerHis
885890895
AsnSerArgLysLys ArgGluIleThrGluLysGlnIleAspAspAsn
900905910
ArgLysPheSerLeuPheAlaGluArgLysTyrGlnThrLeuAsnCys
915 920925
SerValAsnValAsnCysValAsnIleArgCysProLeuArgGlyLeu
930935940
AspSerLysAlaSerLeuIleLeu ArgSerArgLeuTrpAsnSerThr
945950955960
PheLeuGluGluTyrSerLysLeuAsnTyrLeuAspIleLeuMetArg
965 970975
AlaPheIleAspValThrAlaAlaAlaGluAsnIleArgLeuProAsn
980985990
AlaGlyThrGlnValA rgValThrValPheProSerLysThrValAla
99510001005
GlnTyrSerGlyValProTrpTrpIleIleLeuValAlaIleLeuAla
1010 10151020
GlyIleLeuMetLeuAlaLeuLeuValPheIleLeuTrpLysCysGly
1025103010351040
PhePheLysArgSerArg TyrAspAspSerValProArgTyrHisAla
104510501055
ValArgIleArgLysGluGluArgGluIleLysAspGluLysTyrIle
106 010651070
AspAsnLeuGluLysLysGlnTrpIleThrLysTrpAsnArgAsnGlu
107510801085
SerTyrSer
1090
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5499 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 1..5499
(D) OTHER INFORMATION: /product="Human ALPHA 6B"
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 1..3260
(D) OTHER INFORMATION: /note="The sequence of SEQ ID NO:4
is identical to SEQ ID NO:2 between nucleotides 1
and 3260."
(ix) FEATURE:
(A) NAME/KEY: miscfeature
( B) LOCATION: 3261..5499
(D) OTHER INFORMATION: /note="Nucleotides 3261-5499 of
SEQ ID NO:4 are identical to nucleotides 3391-5629
of SEQ ID NO:2. SEQ ID NO:4 has a 130 nucleotide
deletion in relation to SEQ ID NO:2."
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 2924..3325
(D) OTHER INFORMATION: /note="Encompasses the sequence of
the ALPHA 6B cDNA amplified using primers
1156/1157."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GCGCGACCGTCCCGGGGGTGGGGCCGGGCGCAGCGGCGAGAGGAGGCGAAGGTGGCTGCG60
GTAGCAGCAGCGCGGCAGCCTCGGACCCAGCCCGGAGCGCAGGGCGGCCGCTGCAGGTCC120
CCGCTCCCCTCCCC GTGCGTCCGCCCATGGCCGCCGCCGGGCAGCTGTGCTTGCTCTACC180
TGTCGGCGGGGCTCCTGTCCCGGCTCGGCGCAGCCTTCAACTTGGACACTCGGGAGGACA240
ACGTGATCCGGAAATATGGAGACCCCGGGAGCCTCTTCGGCTTCTCGCTGGCCATGCACT 300
GGCAACTGCAGCCCGAGGACAAGCGGCTGTTGCTCGTGGGGGCCCCGCGCGGAGAAGCGC360
TTCCACTGCAGAGAGCCTTCAGAACGGGAGGGCTGTACAGCTGCGACATCACCGCCCGGG420
GGCCATGCACGCGGATCGAGTTTGATAACGATGCTGA CCCCACGTCAGAAAGCAAGGAAG480
ATCAGTGGATGGGGGTCACCGTCCAGAGCCAAGGTCCAGGGGGCAAGGTCGTGACATGTG540
CTCACCGATATGAAAAAAGGCAGCATGTTAATACGAAGCAGGAATCCCGAGACATCTTTG600
GGCGGTGTTATGTC CTGAGTCAGAATCTCAGGATTGAAGACGATATGGATGGGGGAGATT660
GGAGCTTTTGTGATGGGCGATTGAGAGGCCATGAGAAATTTGGCTCTTGCCAGCAAGGTG720
TAGCAGCTACTTTTACTAAAGACTTTCATTACATTGTATTTGGAGCCCCGGGTACTTATA 780
ACTGGAAAGGGATTGTTCGTGTAGAGCAAAAGAATAACACTTTTTTTGACATGAACATCT840
TTGAAGATGGGCCTTATGAAGTTGGTGGAGAGACTGAGCATGATGAAAGTCTCGTTCCTG900
TTCCTGCTAACAGTTACTTAGGTTTTTCTTTGGACTC AGGGAAAGGTATTGTTTCTAAAG960
ATGAGATCACTTTTGTATCTGGTGCTCCCAGAGCCAATCACAGTGGAGCCGTGGTTTTGC1020
TGAAGAGAGACATGAAGTCTGCACATCTCCTCCCTGAGCACATATTCGATGGAGAAGGTC1080
TGGCCTCTTCATTT GGCTATGATGTGGCGGTGATGGACCTCAACAAGGATGGGTGGCAAG1140
ATATAGTTATTGGAGCCCCACAGTATTTTGATAGAGATGGAGAAGTTGGAGGTGCAGTGT1200
ATGTCTACATGAACCAGCAAGGCAGATGGAATAATGTGAAGCCAATTCGTCTTAATGGAA 1260
CCAAAGATTCTATGTTTGGCATTGCAGTAAAAAATATTGGAGATATTAATCAAGATGGCT1320
ACCCAGATATTGCAGTTGGAGCTCCGTATGATGACTTGGGAAAGGTTTTTATCTATCATG1380
GATCTGCAAATGGAATAAATACCAAACCAACACAGGT TCTCAAGGGTATATCACCTTATT1440
TTGGATATTCAATTGCTGGAAACATGGACCTTGATCGAAATTCCTACCCTGATGTTGCTG1500
TTGGTTCCCTCTCAGATTCAGTAACTATTTTCAGATCCCGGCCTGTGATTAATATTCAGA1560
AAACCATCACAGTA ACTCCTAACAGAATTGACCTCCGCCAGAAAACAGCGTGTGGGGCGC1620
CTAGTGGGATATGCCTCCAGGTTAAATCCTGTTTTGAATATACTGCTAACCCCGCTGGTT1680
ATAATCCTTCAATATCAATTGTGGGCACACTTGAAGCTGAAAAAGAAAGAAGAAAATCTG 1740
GGCTATCCTCAAGAGTTCAGTTTCGAAACCAAGGTTCTGAGCCCAAATATACTCAAGAAC1800
TAACTCTGAAGAGGCAGAAACAGAAAGTGTGCATGGAGGAAACCCTGTGGCTACAGGATA1860
ATATCAGAGATAAACTGCGTCCCATTCCCATAACTGC CTCAGTGGAGATCCAAGAGCCAA1920
GCTCTCGTAGGCGAGTGAATTCACTTCCAGAAGTTCTTCCAATTCTGAATTCAGATGAAC1980
CCAAGACAGCTCATATTGATGTTCACTTCTTAAAAGAGGGATGTGGAGACGACAATGTAT2040
GTAACAGCAACCTT AAACTAGAATATAAATTTTGCACCCGAGAAGGAAATCAAGACAAAT2100
TTTCTTATTTACCAATTCAAAAAGGTGTACCAGAACTAGTTCTAAAAGATCAGAAGGATA2160
TTGCTTTAGAAATAACAGTGACAAACAGCCCTTCCAACCCAAGGAATCCCACAAAAGATG 2220
GCGATGACGCCCATGAGGCTAAACTGATTGCAACGTTTCCAGACACTTTAACCTATTCTG2280
CATATAGAGAACTGAGGGCTTTCCCTGAGAAACAGTTGAGTTGTGTTGCCAACCAGAATG2340
GCTCGCAAGCTGACTGTGAGCTCGGAAATCCTTTTAA AAGAAATTCAAATGTCACTTTTT2400
ATTTGGTTTTAAGTACAACTGAAGTCACCTTTGACACCCCATATCTGGATATTAATCTGA2460
AGTTAGAAACAACAAGCAATCAAGATAATTTGGCTCCAATTACAGCTAAAGCAAAAGTGG2520
TTATTGAACTGCTT TTATCGGTCTCGGGAGTTGCTAAACCTTCCCAGGTGTATTTTGGAG2580
GTACAGTTGTTGGCGAGCAAGCTATGAAATCTGAAGATGAAGTGGGAAGTTTAATAGAGT2640
ATGAATTCAGGGTAATAAACTTAGGTAAACCTCTTACAAACCTCGGCACAGCAACCTTGA 2700
ACATTCAGTGGCCAAAAGAAATTAGCAATGGGAAATGGTTGCTTTATTTGGTGAAAGTAG2760
AATCCAAAGGATTGGAAAAGGTAACTTGTGAGCCACAAAAGGAGATAAACTCCCTGAACC2820
TAACGGAGTCTCACAACTCAAGAAAGAAACGGGAAAT TACTGAAAAACAGATAGATGATA2880
ACAGAAAATTTTCTTTATTTGCTGAAAGAAAATACCAGACTCTTAACTGTAGCGTGAACG2940
TGAACTGTGTGAACATCAGATGCCCGCTGCGGGGGCTGGACAGCAAGGCGTCTCTTATTT3000
TGCGCTCGAGGTTA TGGAACAGCACATTTCTAGAGGAATATTCCAAACTGAACTACTTGG3060
ACATTCTCATGCGAGCCTTCATTGATGTGACTGCTGCTGCCGAAAATATCAGGCTGCCAA3120
ATGCAGGCACTCAGGTTCGAGTGACTGTGTTTCCCTCAAAGACTGTAGCTCAGTATTCGG 3180
GAGTACCTTGGTGGATCATCCTAGTGGCTATTCTCGCTGGGATCTTGATGCTTGCTTTAT3240
TAGTGTTTATACTATGGAAGTGTGGATTCTTTAAACGCTCTAGGTACGATGACAGTGTTC3300
CCCGATACCATGCTGTAAGGATCCGGAAAGAAGAGCG AGAGATCAAAGATGAAAAGTATA3360
TTGATAACCTTGAAAAAAAACAGTGGATCACAAAGTGGAACAGAAATGAAAGCTACTCAT3420
AGCGGGGGCCTAAAAAAAAAAAAGCTTCACAGTACCCAAACTGCTTTTTCCAACTCAGAA3480
ATTCAATTTGGATT TAAAAGCCTGCTCAATCCCTGAGGACTGATTTCAGAGTGACTACAC3540
ACAGTACGAACCTACAGTTTTAACTGTGGATATTGTTACGTAGCCTAAGGCTCCTGTTTT3600
GCACAGCCAAATTTAAAACTGTTGGAATGGATTTTTCTTTAACTGCCGTAATTTAACTTT 3660
CTGGGTTGCCTTTGTTTTTGGCGTGGCTGACTTACATCATGTGTTGGGGAAGGGCCTGCC3720
CAGTTGCACTCAGGTGACATCCTCCAGATAGTGTAGCTGAGGAGGCACCTACACTCACCT3780
GCACTAACAGAGTGGCCGTCCTAACCTCGGGCCTGCT GCGCAGACGTCCATCACGTTAGC3840
TGTCCCACATCACAAGACTATGCCATTGGGGTAGTTGTGTTTCAACGGAAAGTGCTGTCT3900
TAAACTAAATGTGCAATAGAAGGTGATGTTGCCATCCTACCGTCTTTTCCTGTTTCCTAG3960
CTGTGTGAATACCT GCTCACGTCAAATGCATACAAGTTTCATTCTCCCTTTCACTAAAAA4020
CACACAGGTGCAACAGACTTGAATGCTAGTTATACTTATTTGTATATGGTATTTATTTTT4080
TCTTTTCTTTACAAACCATTTTGTTATTGACTAACAGGCCAAAGAGTCTCCAGTTTACCC 4140
TTCAGGTTGGTTTAATCAATCAGAATTAGAATTAGAGCATGGGAGGGTCATCACTATGAC4200
CTAAATTATTTACTGCAAAAAGAAAATCTTTATAAATGTACCAGAGAGAGTTGTTTTAAT4260
AACTTATCTATAAACTATAACCTCTCCTTCATGACAG CCTCCACCCCACAACCCAAAAGG4320
TTTAAGAAATAGAATTATAACTGTAAAGATGTTTATTTCAGGCATTGGATATTTTTTACT4380
TTAGAAGCCTGCATAATGTTTCTGGATTTACATACTGTAACATTCAGGAATTCTTGGAGA4440
AGATGGGTTTATTC ACTGAACTCTAGTGCGGTTTACTCACTGCTGCAAATACTGTATATT4500
CAGGACTTGAAAGAAATGGTGAATGCCTATGGAACTAGTGGATCCAAACTGATCCAGTAT4560
AAGACTACTGAATCTGCTACCAAAACAGTTAATCAGTGAGTCGAGTGTTCTATTTTTTGT 4620
TTTGTTTCCTCCCCTATCTGTATTCCCAAAAATTACTTTGGGGCTAATTTAACAAGAACT4680
TTAAATTGTGTTTTAATTGTAAAAATGGCAGGGGGTGGAATTATTACTCTATACATTCAA4740
CAGAGACTGAATAGATATGAAAGCTGATTTTTTTTAA TTACCATGCTTCACAATGTTAAG4800
TTATATGGGGAGCAACAGCAAACAGGTGCTAATTTGTTTTGGATATAGTATAAGCAGTGT4860
CTGTGTTTTGAAAGAATAGAACACAGTTTGTAGTGCCACTGTTGTTTTGGGGGGGGCTTT4920
TTTTCTTTTTCCGG AAAATCCTTAAACCTTAAGATACTAAGGACGTTGTTTTGGTTGTAC4980
TTGGAATTCTTAGTCACAAAATATATTTTGTTTACAAAAATTTCTGTAAAACAGGTTATA5040
ACAGTGTTTAAAGTCTCAGTTTCTTGCTTGGGGAACTTGTGTCCCTAATGTGTTAGATTG 5100
CTAGATTGCTAAGGAGCTGATACTTGACAGTTTTTTAGACCTGTGTTACTAAAAAAAAGA5160
TGAATGTCGGAAAAGGGTGTTGGGAGGGTGGTCAACAAAGAAACAAAGATGTTATGGTGT5220
TTAGACTTATGGTTGTTAAAAATGTCATCTCAAGTCA AGTCACTGGTCTGTTTGCATTTG5280
ATACATTTTTGTACTAACTAGCATTGTAAAATTATTTCATGATTAGAAATTACCTGTGGA5340
TATTTGTATAAAAGTGTGAAATAAATTTTTTATAAAAGTGTTCATTGTTTCGTAACACAG5400
CATTGTATATGTGA AGCAAACTCTAAAATTATAAATGACAACCTGAATTATCTATTTCAT5460
CAAAAAAAAAAAAAAAAAAAACTTTATGGGCACAACTGG5499
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 141 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: 1..141
(D) OTHER INFORMATION: /note="The 141 amino acid sequence
predicted from the nucleic acid product which
results from amplification of the mouse ALPHA 6B
cDNA with primers 1157/1156."
(ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 88..113
(D) OTHER INFORMATION: /note="The putative transmembrane
domain."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: 1..120
(D) OTHER INFORMATION: /note="SEQ ID NO:5 is identical to
SEQ ID NO:7 at amino acid position 1 through 120;
the two sequences diverge at amino acid 121."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ThrLeuAsnCysSerValAsnValArgCysValAsnIleArgCysPro
151015
LeuArgGlyLeuAspSer LysAlaSerLeuValLeuArgSerArgLeu
202530
TrpAsnSerThrPheLeuGluGluTyrSerLysLeuAsnTyrLeuAsp
35 4045
IleLeuLeuArgAlaSerIleAspValThrAlaAlaAlaGlnAsnIle
505560
LysLeuLeuThrAlaGlyThrGlnValArg ValThrValPheProSer
65707580
LysThrValAlaGlnTyrSerGlyValAlaTrpTrpIleIleLeuLeu
85 9095
AlaValLeuAlaGlyIleLeuMetLeuAlaLeuLeuValPheLeuLeu
100105110
TrpLysCysGlyPhePheLysAr gSerArgTyrAspAspSerIlePro
115120125
ArgTyrHisAlaValArgIleArgLysGluGluArgGlu
130135 140
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 426 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 1..426
( D) OTHER INFORMATION: /product="Mouse ALPHA 6B amino
acid sequence in SEQ ID NO:5."
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 262..337
(D) OTHER INFORMATION: /function="Putative transmembrane
region."
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: (342 343)
(D) OTHER INFORMATION: /note="SEQ ID NO:6 is identical to
SEQ ID NO:8 except for 130 nucleotides present in
SEQ ID NO:8 but deleted between nucleotides 342
and 343 of SEQ ID NO:6."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GACTCTTAACTGTAGCGTGAACGTGAGGTGTGTGAACATCAGGTGCCCACTGCGAGGGCT60
GGACAGCAAGGCCTCTCTCGTTCTTCGTTCCAGGTT GTGGAACAGCACATTTCTAGAGGA120
ATATTCCAAACTGAACTACTTGGACATTCTCCTGAGGGCTTCCATAGATGTCACCGCTGC180
TGCTCAGAATATCAAGCTCCTCACCGCCGGCACTCAGGTTCGAGTGACGGTGTTTCCCTC240
AAAGACTGTAGCT CAGTATTCAGGAGTAGCTTGGTGGATCATCCTCCTGGCTGTTCTTGC300
CGGGATTCTGATGCTGGCTCTATTAGTGTTTTTACTGTGGAAGTGTGGATTCTTTAAGCG360
CTCTAGGTACGATGACAGCATTCCCCGATACCATGCGGTGCGGATCCGGAAAGAAGAGC G420
AGAGAT426
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 149 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: 1..149
(D) OTHER INFORMATION: /note="The 149 amino acid sequence
predicted from the product which results from
amplification of the mouse ALPHA 6A cDNA with
primers 1157/1156."
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: 1..120
(D) OTHER INFORMATION: /note="SEQ ID NO:7 is identical to
SEQ ID NO:5 at amino acid positions 1 through 120;
the sequences diverge at amino acid 121."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
ThrLeuAsnCysSerValAsnValArgCysValAsnIleArgCysPro
15 1015
LeuArgGlyLeuAspSerLysAlaSerLeuValLeuArgSerArgLeu
202530
TrpAsnSerThrPheLeuGluGl uTyrSerLysLeuAsnTyrLeuAsp
354045
IleLeuLeuArgAlaSerIleAspValThrAlaAlaAlaGlnAsnIle
5055 60
LysLeuLeuThrAlaGlyThrGlnValArgValThrValPheProSer
65707580
LysThrValAlaGlnTyrSerGlyValAl aTrpTrpIleIleLeuLeu
859095
AlaValLeuAlaGlyIleLeuMetLeuAlaLeuLeuValPheLeuLeu
100 105110
TrpLysCysGlyPhePheLysArgAsnLysLysAspHisTyrAspAla
115120125
ThrTyrHisLysAlaGluIleHisThrGln ProSerAspLysGluArg
130135140
LeuThrSerAspAla
145
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 556 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 1..556
(D) OTHER INFORMATION: /product="Mouse ALPHA 6A amino
acid sequence in SEQ ID NO:7."
/note="SEQ ID NO:8 is the 556 base nucleotide
sequence corresponding to the mouse ALPHA 6A amino
acid sequence SEQ ID NO:7, plus the first 109
nucleotides in the 3'noncoding region."
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 342..472
(D) OTHER INFORMATION: /note="SEQ ID NO:8 is identical to
SEQ ID NO:6 except it has a 130 base insertion
(nucleotides342-472ofSEQIDNO:8) between
nucleotides 352 and 353 of SEQ ID NO:6."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GACTCTTAACTGTAGCGTGAACGTGAGGTGTGTGAACATCAGGTGCCCACTGCGAGGGCT60
GGACAGCAAGGCCTCTCTCGTTCTTCGTTCCAGGTTGTGGAACAGCACATTTCTAGAGGA120
ATATTC CAAACTGAACTACTTGGACATTCTCCTGAGGGCTTCCATAGATGTCACCGCTGC180
TGCTCAGAATATCAAGCTCCTCACCGCCGGCACTCAGGTTCGAGTGACGGTGTTTCCCTC240
AAAGACTGTAGCTCAGTATTCAGGAGTAGCTTGGTGGATCATCCTCCTGGC TGTTCTTGC300
CGGGATTCTGATGCTGGCTCTATTAGTGTTTTTACTGTGGAAGTGTGGCTTCTTCAAGAG360
AAATAAGAAAGATCATTACGATGCCACCTATCACAAGGCTGAGATCCATACTCAGCCGTC420
TGATAAAGAGAGGCTTACTTCCGATGCAT AGTATTGATCTACTTCCATAATTGTGTGGAT480
TCTTTAAGCGCTCTAGGTACGATGACAGCATTCCCCGATACCATGCGGTGCGGATCCGGA540
AAGAAGAGCGAGAGAT556
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 153 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: 1..153
(D) OTHER INFORMATION: /note="SEQ ID NO:9 is the 153
amino acid sequence predicted from the product
which results from amplification of the mouse
ALPHA 3B cDNA with primers 2032/2033."
(ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 108..112
(D) OTHER INFORMATION: /note="The cytoplasmic sequence
CDFFK begins at amino acid position 108."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
AlaArgCysVal TrpLeuGluCysProLeuProAspThrSerAsnIle
151015
ThrAsnValThrValLysAlaArgValTrpAsnSerThrPheIleGlu
202530
AspTyrLysAspPheAspArgValArgValAspGlyTrpAlaThrLeu
354045
PheLeuArgThrSer IleProThrIleAsnMetGluAsnLysThrThr
505560
CysPheSerValAsnIleAspSerLysLeuLeuGluGluLeuProAla
6570 7580
GluIleGluLeuTrpLeuValLeuValAlaValGlyAlaGlyLeuLeu
859095
LeuLeuGlyLeuIle IleIleLeuLeuTrpLysCysAspPhePheLys
100105110
ProThrArgTyrTyrArgIleMetProLysTyrHisAlaValArgIle
115 120125
ArgGluGluAspArgTyrProProProGlySerThrLeuProThrLys
130135140
LysHisTrpValThrSerTrpGlnI le
145150
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 463 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 1..463
(D) OTHER INFORMATION: /product="Mouse ALPHA 3B amino
acid sequence in SEQ ID NO:9."
/note="SEQ ID NO:10 is the 463 base nucleotide
sequence corresponding to the mouse ALPHA 3B amino
acid sequence in SEQ ID NO:9."
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 324..338
(D) OTHER INFORMATION: /product="The cytoplasmic sequence
CDFFK."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GTGCCCGCTGTGTGTGGCTGGAGTGCCCCCTTCCAGACACCTCCAACATTACCAATGTGA60
CCGTGAAAGCACGGGTGTGGAACAGCACCTTCATTGAGGACTACAAAGACTTTGACAGAG120
TCAGG GTAGATGGCTGGGCTACCCTGTTCCTGAGAACCAGCATCCCTACCATCAACATGG180
AGAACAAGACCACATGTTTCTCTGTGAACATTGACTCAAAGCTGTTGGAGGAGCTGCCCG240
CTGAGATTGAGCTGTGGTTGGTGCTTGTGGCCGTGGGTGCTGGGTTGCTG CTGCTGGGGC300
TCATCATCATCCTCTTGTGGAAGTGTGACTTCTTTAAGCCGACCCGCTACTACCGGATTA360
TGCCCAAGTACCATGCAGTGCGTATCCGGGAGGAGGACCGCTACCCACCTCCAGGGAGCA420
CGCTACCCACCAAGAAGCACTGGGTCAC CAGCTGGCAGATTCG463
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /standardname="PCR PRIMER 1157"
/note="Primer corresponds to bp 2918-2937 of the
ALPHA 6A cDNA sequence of SEQ ID NO:2."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GACTCTTAACTGTAGCGTGA20
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /standardname="PCR PRIMER 1156"
/note="The primer corresponds to the complement
of bp 3454- 3473 of the APHA 6A cDNA sequence of
SEQ ID NO:2."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
ATCTCTCGCTCTTCTTTCCG20
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 1..19
(D) OTHER INFORMATION: /standardname="PCR PRIMER 1681"
/note="The primer corresponds to bp 2942-2960 of
the ALPHA 6A cDNA sequence of SEQ ID NO:2."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GAACTGTGTGAACATCAGA19
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /standardname="PCR PRIMER 2002"
/note="The primer corresponds to the complement
of bp 3433- 3452 of the ALPHA 6A cDNA sequence of
SEQ ID NO:2."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
ATCCTTACAGCATGGTATCG20
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /standardname="PCR PRIMER 2032"
/note="The primer corresponds to the hamster
ALPHA 3A cDNA sequence of Tsuji et. al., J. Biol.
Chem., 265:7016-7021 (1990)."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
AAGCCAAATCTGA GACTGTG20
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: miscfeature
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /standardname="PCR PRIMER 2033"
/note="The primer corresponds to the hamster
ALPHA 3A cDNA sequence of Tsuji et al., J. Biol.
Chem., 265:7016-7021 (1990)."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GTAGTATCGGTCCCGAATCT 20
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